NO348288B1 - Polynucleotides encoding anti-CD38 antibodies. - Google Patents

Description

Background of the invention

CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 protein is a bifunctional ectoenzyme that can catalyze the conversion of NAD<+ >to cyclic ACDP-ribose (cADPR) and also hydrolyze cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34<+>-loaded stem cells and lineage-loaded progenitors of lymphoid, erythroid, and myeloid cells. CD38 expression generally remains in the lymphoid lineage with varying levels of expression at different stages of T- and B-cell development.

CD38 is upregulated in many hematopoietic malignancies, and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), B- and T-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, the most primitive, pluripotent stem cells in the hematopoietic system are CD38-. CD38 expression in hematopoietic malignancies and its correlation with disease progression make CD38 an attractive target for antibody therapy.

CD38 has been reported to be involved in Ca<2+> mobilization (M. Morra et al., 1998, FASEB J., 12: 581-592; M. T. Zilber et al., 2000, Proc Natl Acad Sci U S A, 97: 2840-2845) and in signal transduction via tyrosine phosphorylation of numerous signaling molecules, including phospholipase C-γ, ZAP-70, syk and c-cbl in lymphoid and myeloid cells or cell lines (A. Funaro et al., 1993, Eur J Immunol, 23: 2407-2411; M. Morra et al., 1998, FASEB J., 12: 581-592; A. Funaro et al., 1990, J Immunol, 145: 2390-2396; M. Zubiaur et al., 1997, J Immunol, 159: 193-205; S. Deaglio et al., 2003, Blood, 102: 2146-2155; E. Todisco et al., 2000, Blood, 95: 535-542; M. Konopleva et al., 1998, J Immunol, 161: 4702-4708; M.T. Zilber et al., 2000, Proc Natl Acad Sci U S A, 97: 2840-2845; A. Kitanaka et al., 1997, J Immunol, 159: 184-192; A. Kitanaka et al., 1999, J Immunol, 162: 1952-1958; R. Mallone et al., 2001, Int Immunol, 13: 397-409). Based on these observations, CD38 was proposed to be an important signaling molecule in the maturation and activation of lymphoid and myeloid cells during their normal development.

The exact role of CD38 in signal transduction and hematopoiesis is still not fully understood, especially since most of these signal transduction studies have used cell lines that ectopically overexpress CD38 and anti-CD38 monoclonal antibodies, which are non-physiological ligands. Because the CD38 protein has an enzymatic activity that yields cADPR, a molecule that can induce CD<2+> mobilization H. C. Lee et al., 1989, J Biol Chem, 264: 1608-1615; H. C. Lee and R. Aarhus, 1991, Cell Regul, 2: 203-209), it has been proposed that CD38 ligation with monoclonal antibodies triggers Ca<2+> mobilization and signal transduction in lymphocytes by increased production of cADPR (H. C. Lee et al., 1997, Adv Exp Med Biol, 419: 411-419). Against this hypothesis, truncation and point mutation analyses of CD38 protein showed that neither the cytoplasmic tail nor its enzymatic activity is required for signaling mediated by anti-CD38 antibodies (A. Kitanaka et al., 1999, J Immunol, 162: 1952-1958; F. E. Lund et al., 1999, J Immunol, 162: 2693-2702; S. Hoshino et al., 1997, J Immunol, 158, 741-747).

The best evidence for the action of CD38 comes from CD38<-/->-knock-out mice that have a defect in innate immunity and a reduced T-cell-dependent humoral response due to a defect in dendritic cell migration (S. Partida-Sanchez et al., 2004, Immunity, 20: 279-291; S. Partida-Sanchez et al., 2001, Nat Med, 7: 1209-1216). Nevertheless, it is not entirely clear whether the CD38 that functions in mice is identical to that in humans, because the CD38 expression pattern during hematopoiesis differs greatly between humans and mice: a) various immature progenitor stem cells in humans, corresponding to progenitor stem cells in mice, express a high level of CD38 (T. D. Randall et al., 1996, Blood, 87: 4057-4067; R. N. Dagher et al., 1998, Biol Blood Marrow Transplant, 4: 69-74), whereas during human B-cell development, high levels of CD38 expression are found in germinal center B cells and plasma cells (F. M. Uckun, 1990, Blood, 76: 1908-1923; M. Kumagai et al., 1995, J Exp Med, 181: 1101-1110), in mice it is The CD38 expression levels in the corresponding cells low (A. M. Oliver et al., 1997, J Immunol, 158: 1108-1115; A. Ridderstad and D. M. Tarlinton 1998, J Immunol, 160: 4688-4695).

Several anti-human CD38 antibodies with different proliferative properties on different tumor cells and cell lines have been described in the literature. For example, a chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates antibody-mediated cytotoxicity (ADCC) with high efficacy against lymphoma cells in the presence of nucleated peripheral blood effector cells from either MM patients or normal individuals (F. K.

Stevenson et al.1991, Blood, 77: 1071-1079). A CDR-grafted humanized version of the anti-CD38 antibody AT13/5 has been shown to have potent ADCC activity against CD38-positive cell lines (US 09/797941 A1). Human monoclonal anti-CD38 antibodies have been shown to mediate the in vitro killing of CD38-positive cell lines by ADCC and/or complement-dependent cytotoxicity (CDC) (WO2005/103083), and to delay tumor growth in SCID mice with the MM cell line RPMI-8226 (WO 2005/103083 A2). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7 and OKT10, but not IB6, AT1 or AT2, induced proliferation of peripheral blood mononuclear cells (PBMC) from normal individuals (C. M. Ausiello et al. 2000, Tissue Antigens, 56: 539-547).

Some of the prior art antibodies have been shown to be able to induce apoptosis in CD38<+ >B cells. However, they can only do so in the presence of stromal cells or stromal-derived cytokines. An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis of human germinal center (GC) B cells (S. Zupo et al.1994, Eur J Immunol, 24:1218-1222), and to induce proliferation of KG-1 and HL-60 AML cells (M. Konopleva et al. 1998, J Immunol, 161:4702-4708), but induces apoptosis in Jurkat T-lymphoblastic cells (M. Morra et al.1998, FASEB J, 12:581-592). Another anti-CD38 antibody T16 induced apoptosis of immature lymphoid cells and leukemic lymphoblast cells from an ALL patient (M. Kumagai et al.1995, J Exp Med, 181: 1101-1110), and of leukemic myeloblast cells from AML patients (E. Todisco et al. 2000, Blood, 95: 535-542), but T16 induced apoptosis only in the presence of stromal cells or stromal-derived cytokines (IL-7, IL-3, stem cell factor).

On the other hand, certain previously known antibodies induce apoptosis after cross-linking, but are totally free of any apoptotic activity incubated alone (WO 2006/099875).

Because CD38 is an attractive target for antibody therapy for various hematopoietic malignancies, a large number of anti-human CD38 antibodies were generated and screened for high potency in the following three cytotoxic activities against CD38-positive malignant hematopoietic cells: induction of apoptosis, ADCC and CDC. The present disclosure describes novel anti-CD38 antibodies that are capable of killing CD38<+> cells via three distinct cytotoxic mechanisms, namely induction of apoptosis, ADCC and CDC. Notably, the present disclosure describes the first anti-CD38 antibodies that are capable of directly inducing apoptosis of CD38<+> cells, even in the absence of stromal cells or stromal-derived cytokines.

Furthermore, WO 9616990 relates to monoclonal antibodies against CD38 in which foreign CDR regions have been inserted while the framework is of human or primate origin.

Ellis J.H. et al.; "Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma", Journal of Immunology, 1995, vol.155, no.2, pages 925-937 relates to a high-affinity monoclonal antibody against CD38 designated AT13/5.

Summary of the invention

The present invention relates to one or more polynucleotides encoding an anti-CD38 antibody or epitope-binding fragment thereof, wherein the antibody or epitope-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID NO: 66 and a light chain variable region (VL) comprising SEQ ID NO: 62 or 64.

An embodiment of the invention relates to a recombinant vector comprising one or more polynucleotides of the invention.

A further embodiment of the invention relates to a host cell comprising a vector according to the invention.

The present disclosure describes antibodies for specific binding of CD38, and capable of killing CD38<+> cells by apoptosis. While some antibodies according to certain prior art are capable of triggering apoptosis only by crosslinking or crosslinking, but are otherwise devoid of any apoptotic activity, the antibodies according to the disclosure are capable of inducing apoptotic cell death of CD38<+> cells, whether or not they are incubated alone.

Notably, the antibodies described herein are the first anti-CD38 antibodies that have been shown to kill CD38<+ >B cells by all three distinct mechanisms, namely apoptosis, ADCC, and CDC. In a further embodiment, the antibody is capable of killing CD38<+ >B cells by apoptosis even in the absence of stromal cells and stromal-derived cytokines.

The antibodies described herein are capable of specifically killing malignant CD38<+ >B cells including lymphoma, leukemia, and multiple myeloma cells. In certain embodiments, the CD38<+ >B cell is an NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML cell.

Furthermore, the antibodies described herein are capable of killing 24% of Daudi lymphoma cells and/or at least 7% of Ramos lymphocytes and/or 11% of MOLP-8 multiple myeloma cells and/or 36% of SU-DHL-8 lymphocytes and/or 62% of DND-41 leukemia cells and/or 27% of NU-DUL-1 lymphoma cells and/or 9% of JVM-13 leukemia cells and/or 4% of HC-1 leukemia cells by apoptosis in the absence of stromal cells or stromal-derived cytokines.

Antibodies described herein may be polyclonal or monoclonal. Monoclonal anti-CD38 antibodies are preferred. The present disclosure describes murine antibodies selected from 38SB13, 38SB18, 38SB19, 38SB0, 38SB31 and 38SB39, all of which are herein fully characterized with respect to amino acid sequences for both the variable light and heavy regions, identification of their CDRs (complementarity determining regions), identification of their surface amino acids and means for expression in recombinant form.

The present disclosure includes chimeric versions of the murine anti-CD38 monoclonal antibody selected from 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39. Also disclosed are resurfaced or humanized versions of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies in which the surface-exposed residues in the variable framework region of the antibodies, or their epitope-binding fragments, are replaced in both light and heavy chains to more closely resemble known human antibody surfaces. The humanized antibodies and epitope-binding fragments described herein have improved properties in that they are less immunogenic (or completely non-immunogenic) than the murine versions in human subjects to which they are administered. Thus, the different versions of humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies and epitope-binding fragments thereof recognize, in particular, CD38 without being immunogenic to a human.

The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies are fully characterized herein with respect to the respective amino acid sequences for both light and heavy chain variable regions, the DNA sequences for the genes for light and heavy chain variable regions, identification of the complementary determining regions (CDRs), identification of their variable regions in terms of framework surface amino acid residues, and description of means for their expression in recombinant form.

The disclosure also contemplates conjugates between cytotoxic conjugates comprising (1) a cell binding agent that recognizes and binds CD38, and (2) a cytotoxic agent. In the cytotoxic conjugates, the cell binding agent has a high affinity for CD38 and the cytotoxic agent has a high degree of cytotoxicity to cells expressing CD38, such that the cytotoxic conjugates provide effective killing agents.

Preferably, cell binding agents are an anti-CD38 antibody (e.g., 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39) or an epitope-binding fragment thereof, and more particularly a humanized anti-CD38 antibody (e.g., 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39), or an epitope-binding fragment thereof, wherein a cytotoxic agent is covalently linked, directly or via a cleavable or non-cleavable linker, to the antibody or epitope-binding fragment thereof. In greater detail, the cell-binding agent is the humanized 38SB19 antibody, and the cytotoxic agent is a taxol, a maytansinoid, a tomaymycin derivative, a leptomycin derivative, CC-1065 or a CC-1065 analog.

More preferably, the cell binding agent is the humanized anti-CD38 antibody 38SB19, and the cytotoxic agent is a maytansine compound such as DM1 or DM4.

The present disclosure also encompasses the use of fragments of anti-CD38 antibodies that retain the ability to bind CD38. In a further aspect of the disclosure, the use of functional equivalents of anti-CD38 antibodies is contemplated.

The present disclosure also includes a method for inhibiting the growth of a cell expressing CD38. In preferred embodiments, this method for inhibiting the growth of cells expressing CD38 occurs in vivo, resulting in cell death, although in vitro and ex vivo uses are also included.

The present disclosure also describes a pharmaceutical composition comprising an anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agent conjugate, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the therapeutic agent comprises a second therapeutic agent. This second therapeutic agent may be selected from the group comprising antagonists of epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF), or an antagonist of a receptor for epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF) including HER2 receptor, HER3 receptor, c-Met and other receptor tyrosine kinases. This second therapeutic agent may also be selected from the group comprising antibodies targeting cluster of differentiation (CD) antigens including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. This second therapeutic agent may also be selected from the group of chemotherapeutics or immunomodulatory agents.

The present disclosure further describes the antibody or conjugate for use in treating a subject with a cancer or an inflammatory disease including autoimmune disease using the therapeutic compositions. In certain embodiments, the cancer is selected from the group consisting of NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL and CML. In yet another embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranous proliferative glomerulonephritis, Sjogren's disease, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, neuromyelitis optica, Graves' dysthyroid disease, bullous pemphigoid, membranous proliferative glomerulonephritis, Churg-Strauss syndrome, and asthma. In preferred embodiments, the cytotoxic conjugate comprises an anti-CD38 antibody and a cytotoxic agent. In more preferred embodiments, the cytotoxic conjugate comprises a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody DM1 conjugate, humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody DM4, or a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody taxane conjugate, and the conjugate is administered together with pharmaceutically acceptable carriers or excipients.

It is further described that anti-CD38 antibodies can be used to detect the CD38 protein in a biological sample. The antibodies can also be used to determine CD38 levels in tumor tissue.

The present disclosure also describes a kit comprising an anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agent conjugate and instructions for use. In a preferred embodiment, the anti-CD38 antibodies are the humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies, the cytotoxic agent is a maytansine compound such as DM1 or DM4, a tomaymycin derivative, a leptomycin derivative or a taxane, and instructions for use of the conjugates in treating a subject with cancer. The kit may also include components necessary for the preparation of a pharmaceutically acceptable formulation, such as a diluent if the conjugate is in a lyophilized state, or in concentrated form, and for administering the formulation.

Brief description of the figures

Figure 1A shows a FACS analysis of the specific binding of purified murine anti-CD38 antibodies, 38SB12, 38SB18, 38SB19, 38 and 300-19 cells expressing human CD38 and CD38-positive Ramos lymphoma cells;

Figure 1B shows a FACS analysis of the specific binding of purified murine anti-CD38 antibodies, 38SB30, 38SB31, 38SB39 and control anti-CD38 antibody AT13/15 to 300-19 cells expressing human CD38 and CD38-positive Ramos lymphoma cells; Figure 2 shows the binding titration curves for 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 established with Ramos cells;

Figure 3 shows FACS dot plot (FL4-H; TO-PRO-3 staining; y-axis and FL1-H; Annexin V-FITC staining; x-axis) of Ramos cells undergoing apoptosis after incubation with 38SB13, 38SB19 or AT13/5 (10 nM) for 24 h;

Figure 4A shows the mean percentages of Ramos cells undergoing apoptosis after a 24-hour incubation with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, 38SB39, 38SB7, 38SB23, IB4, AT13/5, OKT10, or SUN-4B7. The mean percentage of annexin V-positive cells (y-axis: includes both TO-PRO-3-positive and -negative cells) from duplicate samples was plotted;

Figure 4B shows the mean percentages of Daudi cells undergoing apoptosis after a 24-hour incubation with the same set of antibodies as indicated in Figure 4A; Figure 4C shows the mean percentages of Molp-8 cells undergoing apoptosis after a 24-hour incubation with the same set of antibodies as indicated in Figure 4A;

Figure 5A shows a diagram of the human expression vector used to express hu38SB19-LC;

Figure 5B shows a diagram of the human expression vector used to express hu38SB19-HC;

Figure 5C shows a diagram of the human expression vector used to express both hu38SB19LC and hu38SB19HC;

Figure 6A shows ADCC activities mediated by antibodies ch38SB13, ch38SB18 and ch38SB19 against Ramos cells;

Figure 6B shows ADCC activities mediated by antibodies ch38SB30, ch38SB31 and ch38SB39 against Ramos cells;

Figure 7A shows ADCC activities mediated by antibodies ch38SB18, ch38SB19, ch38SB31, and non-binding, chimeric human IgG1 control antibody against LP-1 multiple myeloma cells;

Figure 7B compares ADCC activities mediated by the antibodies ch38SB19 and murine 38SB19 against Daudi cells;

Figure 8A shows ADCC activities mediated by the ch38SB19 antibody and by the non-binding, chimeric human IgG1 control antibody against NALM-6 B-ALL cells;

Figure 8B shows ADCC activities mediated by the ch38SB19 antibody and by the non-binding, chimeric human IgG1 control antibody against MOLT-4 T-ALL cells;

Figure 9A shows CDC activities mediated by antibodies ch38SB13, ch38SB18, ch38SB19, ch38SB30 and ch38SB39 against Raji-IMG cells;

Figure 9B shows CDC activities mediated by antibodies ch38SB19 and ch38SB31 against Raji-IMG cells;

Figure 10 shows CDC activities mediated by the antibodies ch38SB18, ch28SB19, ch28SB31 and by the non-binding, chimeric human IgG1 control antibody, against LP-1 multiple myeloma cells;

Figure 11A shows CDC activities mediated by antibodies ch38SB13, ch38SB19 and ch38SB39 against Daudi cells;

Figure 11B shows CDC activities mediated by antibodies ch38SB18 and ch38SB30 against Daudi cells;

Figure 11C shows CDC activities mediated by antibodies ch38SB19 and ch38SB31 against Daudi cells;

Figure 12A shows the binding titration curves for ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 for binding to Ramos cells;

Figure 12B shows the binding curves comparing ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.00 for ability to compete with binding of biotinylated murine 38SB19 antibody to Ramos cells;

Figure 13 shows the mean percentages of Daudi cells undergoing apoptosis after 24-hour incubation with ch38SB19, hu38SB19 v1.00 or hu38SB19 v1.20 antibody;

Figure 14 shows ADCC activities mediated by the antibodies ch38SB19, hu38SB19 v1.00, hu38SB19 v1.20, and by non-binding, chimeric, human IgG1 control antibody against LP-1 multiple myeloma cells;

Figure 15A shows CDC activities mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 against Raji-IMG lymphoma cells;

Figure 15B shows CDC activities mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 against LP-1 multiple myeloma cells;

Figure 15C shows CDC activities mediated by antibodies ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 against DND-41 T-cell acute lymphoblastic leukemia cells;

Figure 16 shows the mean percentages of Annexin-V positive cells after 24 hours of incubation with hu38SB19 v1.00 antibody for SU-DHL-8 diffuse large B-cell lymphoma cells, NU-DUL-1 B-cell lymphoma cells, DND-41 T-cell acute lymphoblastic leukemia cells, JVM-13 B-cell chronic lymphocytic leukemia cells, and HC-1 hairy cell leukemia cells;

Figure 17 shows the percentage survival of SCID mice bearing established disseminated human Ramos tumors. Mice were treated with murine 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody or PBS as indicated;

Figure 18 shows the percentage survival of SCID mice with established disseminated human Daudi tumors. Mice were treated with hu38SB19 or mu38SB19 antibody or PBS as indicated;

Figure 19 shows the mean tumor volume of SCID mice bearing NCI-H929 multiple myeloma xenograft tumors. Mice were treated with hu38SB19, a non-binding control IgG1 antibody, or PBS as indicated; and

Figure 20 shows the mean tumor volume of SCID mice bearing MOLP-8 multiple myeloma xenograft tumors. The mice were treated with hu38SB19, mu38SB19, a non-binding control IgG1 antibody, and PBS as indicated.

Detailed description of the invention

The invention is defined in the accompanying set of claims.

The present invention relates to one or more polynucleotides encoding an anti-CD38 antibody or epitope-binding fragment thereof, wherein the antibody or epitope-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID NO: 66 and a light chain variable region (VL) comprising SEQ ID NO: 62 or 64.

An embodiment of the invention relates to a recombinant vector comprising one or more polynucleotides of the invention.

A further embodiment of the invention relates to a host cell comprising a vector according to the invention.

Antibodies are described that are capable of specifically binding CD38. In particular, novel antibodies are described that specifically bind to CD38 on the cell surface and kill CD38<+> cells by apoptosis, by antibody-dependent cytotoxicity (ADCC) and by complement-dependent cytotoxicity (CDC). Thus, the first anti-CD38 antibodies are described that are capable of killing a CD38<+> cell by three different mechanisms.

Antibodies capable of binding CD38 and inducing apoptotic cell death in CD38<+> cells have been previously described (M. Kumagai et al., 1995, J Exp Med, 181: 1101-1110; E. Todisco et al. 2000, Blood, 95: 535-542), but the antibodies described herein are the first to demonstrate apoptotic activity in the absence of stromal cells or stromal-derived cytokines. The term “stroma,” as used herein, refers to the nonmalignant supporting tissue of a tumor that includes connective tissue, blood vessels, and inflammatory cells. Stromal cells produce growth factors and other substances, including cytokines, that can influence the behavior of cancer cells. The term "cytokine" as used herein refers to small, secreted proteins (e.g., IL-1, IL-2, IL-4, IL-5, and IL-6, IFNg, IL-3, IL-7, and GM-CSF) that mediate and regulate immunity, inflammation, and hematopoiesis. It has been shown herein that the antibodies of the prior art are unable to induce apoptotic cell death in the absence of stromal cells or stromal-derived cytokines. In contrast, anti-CD38 antibodies of the disclosure, under the same conditions, exhibit apoptotic activities.

In yet another aspect, the antibodies of the disclosure are capable of binding CD38 protein with a kD of 3 x 10<-9 >M or less.

The term "CD38", as used herein, refers to a type II transmembrane protein comprising, for example, an amino acid sequence as in Genban accession no. NP_001766. A "CD38<+> cell" is a cell that expresses the CD38 protein. Preferably, the CD38<+> cell is a mammalian cell.

The CD38<+> cell is preferably a malignant cell. The CD38<+> cell may be a B cell.

Preferably, the CD38<+> cell is a tumor cell derived from a hematopoietic malignancy. In a preferred embodiment, the CD38<+> cell is a lymphoma cell, a leukemia cell, or a multiple myeloma cell. In a further embodiment, the CD38<+> cell is an NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML cell.

Anti-CD38 antibodies are described capable of killing at least 24% of Daudi lymphoma cells in the absence of stromal cells or stromal-derived cytokines. In another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 7% of Ramos lymphoma cells in the absence of stromal cells or stromal-derived cytokines. In another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 11% of MOLP-8 multiple myeloma cells in the absence of stromal cells or stromal-derived cytokines. In another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 36% of SU-DHL-8 lymphoma cells in the absence of stromal cells or stromal-derived cytokines. In yet another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 27% of NU-DUL-1 lymphoma cells in the absence of stromal cells or stromal-derived cytokines. In yet another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 62% of DND-41 leukemia cells in the absence of stromal cells or stromal-derived cytokines. In yet another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 9% of JVM-13 leukemia cells in the absence of stromal cells or stromal-derived cytokines. In yet another embodiment, the anti-CD38 antibodies of the disclosure are capable of killing at least 4% of HC-1 leukemia cells in the absence of stromal cells or stromal-derived cytokines.

Antibodies

The term "antibody" is used herein in its broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies and antibody fragments. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunization of an animal with the antigen or an antigen-encoding nucleic acid.

A typical IgG antibody consists of two identical heavy chains and two identical light chains joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called “complementarity determining regions” (“CDRs”) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen. They are usually designated as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The more highly conserved parts of the variable regions are called “framework regions”.

As used herein, “VH” or “VH” refers to the variable regions of an immunoglobulin heavy chain of an antibody including the heavy chain of an Fv, scFv, dsFv, Fab, Fab’, or F(ab’)2 fragment. Reference to “VL” or “VL” refers to the variable region of the immunoglobulin light chain of an antibody including the light chain of an Fv, scFv, dsFv, Fab, Fab’, or F(ab’)2 fragment.

A “polyclonal antibody” is an antibody that was produced among or in the presence of one or more other, unidentified antibodies. Generally, polyclonal antibodies are produced from a B lymphocyte in the presence of other B lymphocytes that produce non-identical antibodies. Typically, polyclonal antibodies are obtained directly from an immunized animal.

A “monoclonal antibody”, as used herein, is an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies that make up that population are substantially identical, except for possible naturally occurring mutations that may be present in minor amounts. These antibodies are directed against a single epitope and are therefore highly specific.

An “epitope” is the site on the antigen to which the antibody binds. If the antigen is a polymer such as a protein or polysaccharide, the epitope may be formed by adjacent residues or by nonadjacent residues brought into proximity by folding of an antigenic polymer. In proteins, epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by nonadjacent amino acids are typically lost upon said exposure.

As used herein, the term "KD" refers to the dissociation constant for a particular antibody/antigen interaction.

The present disclosure proceeds from novel murine anti-CD38 antibodies, herein 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39, which are fully characterized with respect to the amino acid sequences of both light and heavy chains, identification of the CDRs, identification of the surface amino acids and means for their expression in recombinant form. The primary amino acid and DNA sequences of antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 light and heavy chains, and humanized versions, are described herein.

The hybridoma cell lines producing the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 murine anti-CD38 antibodies have been deposited with the ATCC (10801 University Bld., Manassas, VA, 20110-2209, USA) on June 21, 2006 under accession numbers PTA-7667, PTA-7669, PTA-7670, PTA-7666, PTA-7668, and PTA-7671, respectively.

The present disclosure is not limited to antibodies and fragments comprising these sequences. Thus, antibodies and antibody fragments are described which may differ from antibodies 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 or humanized derivatives in the amino acid sequence of their framework CDRs, light and heavy chains.

The present disclosure provides antibodies or epitope-binding fragments thereof comprising one or more CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36. As disclosed, the antibodies comprise at least one heavy chain and at least one light chain, and wherein the heavy chain comprises three sequential CDRs having amino acid sequences selected from the group consisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32 and 33, and said light chain comprises three sequential CDRs with amino acid sequences selected from the group consisting of SEQ ID NOS: 4, 5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35 and 36.

Preferably, the antibodies of the disclosure comprise three CDRs with amino acid sequences selected from the group of SEQ ID NOS: 13, 14, 15, 16, 17 and 18. In yet another, more preferred embodiment, there is provided a 38SB19 antibody comprising at least one heavy chain and at least one light chain, and wherein the heavy chain comprises three sequential CDRs with amino acid sequences consisting of SEQ ID NOS: 13, 14 and 15, and light chains comprising three sequential CDRs with amino acid sequences consisting of SEQ ID NOS: 16, 17 and 18.

Preferably, the anti-CD38 antibodies of the disclosure comprise a VL having a sequence according to SEQ ID NO: 42. More preferably, there is provided a 38SB19 antibody comprising a VL having an amino acid sequence consisting of SEQ ID NO: 42.

Antibodies comprising a VH with an amino acid sequence according to SEQ ID NO: 54 are also provided. In a more preferred embodiment, a 38SB19 antibody comprising a VH with an amino acid sequence consisting of SEQ ID NO: 54 is provided.

Chimeric and humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies

As used herein, a “chimeric antibody” is an antibody in which the constant region, or a portion thereof, has been altered, replaced, or exchanged so that the variable region is linked to a constant region of another species, or belongs to another antibody class or subclass. “Chimeric antibody” also refers to an antibody in which the variable region, or a portion thereof, has been altered, replaced, or exchanged so that the constant region is linked to a variable region of another species, or belongs to another antibody class or subclass. Methods for preparing chimeric antibodies are well known in the art, see, for example, Morrison, 1985, Science, 229: 1202; Oi et al., 1986, BioTechniques, 4: 214; Gillies et al., 1989, J. Immunol. Methods, 125: 191-202; U.S. Patent No. 5,807,715; 4,816,567 and 4,816,397.

In one embodiment, chimeric versions of 38SB19 are provided. In particular, said chimeric versions contain at least one human constant region. In a more preferred embodiment, said human constant region is the human IgG1/kappa constant region.

The term “humanized antibody”, as used herein, refers to a chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. The purpose of humanization is to reduce the immunogenicity of a xenogeneic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted for non-rejection by other mammals, can be prepared using various technologies such as “resurfacing” and CDR grafting. As used herein, resurfacing technology uses a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surface of antibody variable regions to resemble the surfaces of known antibodies in the target host. CDR grafting technology involves replacing the complementarity determining regions of, for example, a mouse antibody with a human framework region, see, for example, WO 92/22653.

Humanized, chimeric antibodies preferably have constant regions and variable regions other than the complementarity determining regions (CDRs) derived essentially or exclusively from the corresponding human antibody regions and CDRs derived essentially or exclusively from a mammal other than a human.

Strategies and methods for resurfacing antibodies, and or methods for reducing the immunogenicity of antibodies in a different host, are described in US 5639 641. Briefly, in a preferred method

(1) alignments of a fusion of antibody heavy and light chain variable regions generated to provide a set of heavy and light chain variable region framework surface-exposed positions where the alignment position of all variable regions is at least about 98% identical;

(2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof);

(3) a set of heavy and light variable region framework surface exposed amino acid residues that are nearly identical to the set of rodent surface exposed amino acid residues has been identified;

(4) the set of heavy and light chain variable region framework surface exposed amino acid residues as defined in step (2) is replaced with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5Å of any atom of any residue in the complementarity determining regions of the rodent antibody; and

(5) the humanized rodent antibody with binding specificity is prepared.

Antibodies can be humanized using a number of other techniques including CDR grafting (EP 0239 400, WO 91/09967, US Patent Nos. 5,530,101 and 5,585,089) “veneering” or “resurfacing” (EP 0592 106, EP 0519596, Padlan E. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. et al., 1994, Protein Engineering, 7(6): 805-814; Roguska M.A. et al., 1994, PNAS, 91: 969-973) and chain shuffling (US Patent No. 5,565,332). Human antibodies can be produced by a number of methods well known in the art, including phage display methods, see also US 4,444,887, 4,716,111, 5,545,806 and 5,814,318, as well as WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.

The present disclosure describes humanized antibodies that recognize CD38 and kill CD38<+> cells by apoptosis, ADCC and/or CDC. In a further embodiment, the humanized antibodies or epitope-binding fragments thereof are capable of killing CD38<+> cells by apoptosis even in the absence of stromal cells or stromal-derived cytokines.

A preferred embodiment of such a humanized antibody is a 38SB19 antibody.

In a further preferred embodiment, resurfaced or humanized versions of the 38SB19 antibodies are provided in which the surface-exposed residues of the antibody or fragments thereof are replaced in both the light and heavy chains to more closely resemble known human antibody surfaces. The humanized 38SB19 antibodies have improved properties. For example, humanized 38SB19 antibodies or epitope-binding fragments thereof specifically recognize the CD38 protein. The humanized 38SB19 antibodies have the additional ability to kill a CD38<+> cell, by apoptosis, ADCC and CDC.

The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies are also fully characterized herein with respect to their respective amino acid sequences for both light and heavy chain variable regions, the DNA sequences of the light and heavy chain variable region genes, identification of the CDRs, identification of their surface amino acids, descriptions of a means for their expression in recombinant form.

The CDRs of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies have been identified by modeling and their molecular structures predicted. Once again, while the CDRs are important for epitope recognition, they are not essential to the antibodies and fragments. Accordingly, antibodies and fragments having improved properties produced by, for example, affinity maturation of an antibody are provided.

The heavy chain and light chain variable region sequences of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies and the sequences of their CDRs were not previously known and are disclosed herein. Such information can be used to produce humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies. These humanized anti-CD38 antibodies, or their derivatives, can also be used as cell binding agents.

Thus, in one embodiment, a humanized version of 38SB19 is provided comprising at least one heavy chain and at least one light chain, and wherein said heavy chain comprises three sequential, complementarity determining regions with amino acid sequences represented by SEQ ID NOS: 13, 14 and 15, and wherein said light chain comprises three sequential, complementarity determining regions with amino acid sequences represented by SEQ ID NOS: 16, 17 and 18.

In a preferred embodiment, a humanized 38SB19 antibody is provided comprising a VH having an amino acid sequence represented by SEQ ID NO: 66

In a preferred embodiment, a humanized 38SB19 antibody is provided that comprises a VL having an amino acid sequence selected from the group consisting of SEQ ID NOS: 62 and 64. The humanized 38SB19 antibodies may also include substitutions in light and/or heavy chain amino acid residues at one or more positions defined by the gray residues in Tables 1A and 1B that represent the murine surface framework residues that are changed from the original murine residue to the corresponding framework surface residue in the human antibody 28E4. The dashed (*) residues in Table 1B correspond to the murine back mutations in the humanized 38SB19 heavy chain variant (SEQ ID NO: 69). The residues for back mutations are proximal to the CDRs and were selected as described in US 5,639,641 or in analogy to the selection of residues that in previous humanization attempts resulted in a reduction in antigen binding affinity (Roguska et al., 1996, US publ. ;2003/0235582 and 2005/0118183). ;;Similarly, the humanized 38SB13, 38SB18, 38SB30, 38SB31 and 38SB39 antibodies and epitope-binding fragments thereof may also include substitution in light and/or heavy chain amino acid residues. ;;Polynucleotides, Vectors and Host Cells ;The present invention provides nucleic acids encoding anti-CD38 antibodies described herein. In one embodiment, the nucleic acid molecule encodes a heavy and/or a light chain of an anti-CD38 immunoglobulin. In a preferred embodiment, a single nucleic acid encodes a heavy chain of an anti-CD38 immunoglobulin and a second nucleic acid molecule encodes the light chain of an anti-CD38 immunoglobulin. ;;There are also provided polynucleotides encoding polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72. The polynucleotides of the invention is selected from the group consisting of SEQ ID NOS: 62, 64 and 66. ;;The present invention provides vectors comprising polynucleotides of the invention. In one embodiment, the vector contains a polynucleotide encoding a heavy chain of an anti-CD38 immunoglobulin. In another embodiment, the polynucleotide encodes the light chain of an anti-CD38 immunoglobulin. The invention also provides vectors comprising polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments and probes thereof. ;;To express the heavy and/or light chain of anti-CD38 antibodies of the disclosure, the polynucleotides encoding said heavy and/or light chain are inserted into expression vectors such that the genes are operably linked to transcriptional and translational sequences. Expression vectors include plasmids, YACs, cosmids, retroviruses, EBV-derived episomes and all other vectors that the skilled person will know to be appropriate for ensuring expression of said heavy and/or light chains. ;The skilled person will realize that the polynucleotides encoding the heavy and light chains can be cloned into different vectors or into the same vector. In a preferred embodiment, these polynucleotides are cloned into the same vector. ;;The polynucleotides of the invention, and vectors comprising these molecules, can be used for transforming a suitable mammalian host cell. The transformation can be done by any known method for introducing polynucleotides into a host cell. Such methods are well known to those skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, biolistic injection and direct microinjection of DNA into the nucleus. ;;Antibody Fragments ;As used herein, “antibody fragments” include any portion of an antibody that retains the ability to bind to the epitope recognized by the full-length antibody, generally referred to as “epitope-binding fragments”. Examples of antibody fragments include, but are not limited to, Fab, Fab’ and F(ab’)2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH region. Epitope-binding fragments, including single chain antibodies, may comprise the variable region(s) alone or in combination with all or part of the following: hinge region, CH1, CH2 and CH3 domains. ;;Such fragments may contain one or both Fab fragments or the F(ab’)2 fragment. Preferably, antibody fragments contain all six CDRs of the entire antibody, although fragments containing fewer than all such regions as three, four or five CDRs are also functional. Furthermore, the fragments may be per se or combine members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE, and subclasses thereof. ;;Fab and F(ab')2 fragments can be prepared by proteolytic cleavage, using enzymes such as papain (Fab fragments) and pepsin (F(ab')2 fragments). ;;"Single chain FVer" ("scFver") fragments are epitope-binding fragments that contain at least one fragment of an antibody heavy chain variable region (VH) linked to at least one fragment of an antibody light chain variable region (VL). The linker may be a short, flexible peptide selected to ensure that the correct three-dimensional folding of the VL and VH regions occurs once they are joined to maintain the target binding specificity of the whole antibody from which the single chain antibody fragment is derived. The carboxyl terminus of the VL or VH sequence may be covalently linked by a linker to the amino acid terminus of a complementary VL or VH sequence. ;;Single chain antibody fragments contain amino acid sequences with at least one of the variable or complementarity determining regions (CDRs) of the whole antibodies as described in the specification, but lack some or all of the constant domains of these antibodies. These constant domains are not required for antigen binding, but constitute a major portion of the structure of the whole antibodies. Single chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing part or all of a constant domain. For example, single chain antibody fragments tend to be free of unwanted interactions between biological molecules and the heavy chain constant region, or other unwanted biological activity. In addition, single chain antibody fragments are significantly smaller than whole antibodies, and therefore may have a greater capillary permeability than whole antibodies, allowing single chain antibody fragments to localize and bind to target antigen binding sites more efficiently. Furthermore, antibody fragments can be produced on a relatively small scale in prokaryotic cells, which facilitates their production. Furthermore, the relatively small size of single chain antibody fragments makes them less likely to elicit an immune response in a recipient compared to whole antibodies. ;;Single chain antibody fragments can be generated by molecular cloning, antibody phage display libraries, or similar techniques well known to those skilled in the art. These proteins can be produced, for example, in eukaryotic cells or prokaryotic cells, including bacteria. The epitope-binding fragments can also be generated using various phage display methods, as is well known in the art. In phage display methods, functional antibody domains are displayed on the surface of the phage particle carrying the polynucleotide sequences encoding them. In particular, such a phage can be used to display epitope-binding domains expressed from a repertoire or a combinatorial antibody library (e.g., human or murine). Phage expressing an epitope-binding domain that binds the antigen of interest can be selected or identified with antigen, for example, using labeled antigen bound to or captured on a solid surface or a bead. Phage used in these methods is typically a filamentous phage including fd and M13-binding domains expressed from phage with Fab, Fv, or disulfide-stabilized F antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare the epitope binding fragments include those described in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50; Ames et al., 1995, J. Immunol. Methods, 184: 177-186; ; Kettleborough et al., 1994, Eur. J. Immunol., 24: 952-958; Persic et al., 1997, Gene, 187: 9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280; WO/1992/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US patent no. 5,698,426; 5223 409; 5403 484; 5580 717; 5427 908; 5750 753; 5821 047; 5571 698; 5427 908; 5516 637; 5,780,225; 5658 727; 5733 743 and 5969 108. ;After phage selection, the regions of the phage encoding the fragments can be isolated and used to generate the epitope-binding fragments via expression in a selected host, including mammalian cells, insect cells, plant cells, yeast and bacteria, using recombinant DNA technology, for example as described in detail below. For example, techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be used using methods that are well known and as described in WO 92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; Sawai et al., 1995, AJRI, 34: 26-34; and Better et al., 1988, Science, 240:1041-1043. Examples of techniques that can be used to prepare single chain Fver and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology, 203:46-88; Shu et al., 1993, PNAS, 90:7995-7999; Skerra et al., 1988, Science, 240:1038-1040. ;;Cytotoxic conjugates are also described. These cytotoxic conjugates comprise two primary components, a cell binding agent and a cytotoxic agent. ;;As used herein, the term “cell binding agent” refers to an agent that specifically recognizes and binds the CD38 proteins on the cell surface. In one embodiment, the cell binding agent specifically recognizes CD38, allowing the conjugates to act in a targeted manner, with little side effects resulting from non-specific binding. ;;In another embodiment, the cell binding agent also specifically recognizes the CD38 protein, such that the conjugates will be in contact with the target cell for a sufficient period of time to allow the cytotoxic drug portion of the conjugate to act on the cell, and/or to allow the conjugates sufficient time to be internalized by the cell. ;;In a preferred embodiment, the cytotoxic conjugates comprise an anti-CD38 antibody as the cell binding agent antibody38SB19-, antibody is capable of specifically recognizing CD38 and directing the cytotoxic agent to an abnormal cell or tissue, such as cancer cells, in a targeted manner. ;;The second component of the cytotoxic conjugates is a cytotoxic agent. ;The term “cytotoxic agent,” as used herein, refers to a substance that reduces or blocks the function, or growth, of cells and/or causes destruction of cells. ;In preferred embodiments, the cytotoxic agent is a small drug, a prodrug, a taxoid, a maytansinoid such as DM1 or DM4, a tomaymycin derivative, a leptomycin derivative, CC-1065, or a CC-1065 analog. In preferred embodiments, the cell binding agent is covalently attached, directly or via a cleavable or non-cleavable linker, to the cytotoxic agent. ;;The cell binding agents, cytotoxic agents, and linkers are discussed in greater detail below. ;;Cell Binding Agents ;The effectiveness of the compounds of the disclosure as therapeutic agents depends on the careful selection of a suitable cell binding agent. Cell binding agents may be of any type known today, or that becomes known, and include peptides and non-peptides. The cell binding agent may be any compound that can bind a cell, either specifically or non-specifically. In general, these may be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (such as transferrin), or any other cell binding molecule or such substance. ;;More specific examples of cell binding agents that may be used include: ;a) polyclonal antibodies; ;;b) monoclonal antibodies; ;;c) fragments of antibodies such as Fab, Fab' and F(ab')2, Fv (Parham, 1983, J. ;Immunol., 131: 2895-2902; Spring et al., 1974, J. Immunol., 113: 470-478; Nisonoff et al., 1960, Arch. Biochem. Biophys., 89: 230-244). ;;In particular, an anti-CD38 monoclonal antibody selected from 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 may be used as a cell binding agent. Likewise, said cell binding agent may be a chimeric version of one of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 monoclonal antibodies. Preferably, a humanized anti-CD38 antibody is used as a cell binding agent. More particularly, the humanized anti-CD38 antibody is selected from humanized or resurfaced 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies. ;Cytotoxic Agents ;In a further embodiment, the humanized antibody may be conjugated to a drug such as a maytansinoid, to provide a prodrug with specific cytotoxicity against antigen-expressing cells by targeting the drug to the CD38 protein. ;Cytotoxic conjugates comprising such antibodies and a low and high toxicity drug (e.g., maytansinoids, taxanes, tomaymycin derivatives, leptomycin derivatives, and CC-1065 analogs) may be used as a therapeutic for the treatment of tumors such as lymphoma, leukemia, and multiple myeloma. ;The cytotoxic agent used in the cytotoxic conjugate may be any compound that results in the death of a cell, or induces cell death, or in some way reduces cell viability. Preferred cytotoxic agents include, for example, maytansinoids and maytansinoid analogs, taxoids, tomaymycin derivatives, leptomycin derivatives, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, as defined below. These cytotoxic agents are conjugated to the antibodies, antibody fragments, functional equivalents, improved antibodies and their analogs as described herein. ;;The cytotoxic conjugates can be prepared by in vitro methods. To link a drug or prodrug to the antibody, a linker group is used. Suitable linker groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Preferred linker groups are disulfide groups and thioether groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between the antibody and the drug or prodrug. ;;Maytansinoids ;Among the cytotoxic agents that can be used according to the disclosure to provide a cytotoxic conjugate are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells. ;;Examples of suitable maytansinol analogs include those with a modified aromatic ring, and those with modifications at other positions. Such suitable maytansinoids are described in US 4,424,219, 4,256,746, 4,294,757, 4,307,016, 4,313,943, 4,315,929, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,450,254, 4,322,348, 4,371,533, ;6,333,410, 5,475,092, 5,585,499 and 5,846,545. ;;Specific examples of suitable analogs of maytansinol with a modified aromatic ring include: ;;(1) C-19-dechloro (US 4256 746) (prepared by LAH reduction of ansamytocin P2); ;;(2) C-20-hydroxy (or C-20-demethyl) /- C-19-dechloro (US 4361 650 and ;4 307 016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and ;;(3) C-20-demethoxy, C-20-acyloxy (-OCOR), /- dechloro (US 4294 757) (prepared by acylation using acyl chlorides). ;;Specific examples of suitable analogs of maytansinol with modifications at other positions include: ;;(1) C-9-SH (US 4424 219) (prepared by reacting maytansinol with H2S or P2S5); ;;(2) C-14-alkoxymethyl (demethoxy/CH2OR) (US 4331 598); ;;(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US ;4 450 254) (prepared from Nocardia); ;;(4) C-15-hydroxy/acyloxy (US 4364 866) (prepared by converting maytansinol with Streptomyces); ;;(5) C-15-methoxy (US 4313 946 and 4315 929) (isolated from Trewia nudiflora); ;;(6) C-18-N-demethyl (US 4362 663 and 4322 348) (prepared by demethylation of maytansinol with Streptomyces); and ;(7) 4,5-deoxy (US 4371 533) (prepared by titanium trichloride/LAH reduction of maytansinol). ;;In a preferred embodiment, the cytotoxic conjugates utilize the thiol-containing maytansinoid (DM1), formally called N2’-deacetyl-N2’-(3-mercapto-1-oxopropyl)-maytansine, as the cytotoxic agent. DM1 is represented by the following structural formula (I): ;;;;;;In another preferred embodiment, the cytotoxic conjugates utilize the thiol-containing maytansinoid N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine as the cytotoxic agent. DM4 is represented by the following structural formula (II): ;;;;;;In further embodiments, other maytansines including thiol- and disulfide-containing maytansinoids bearing a mono- or dialkyl substitution on the carbon atom bearing the sulfur atom may be utilized. These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl, an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group where the carbon atom of the acyl group bearing the thiol functionality has one or two substituents, and where the substituent is CH3, CH2H5, straight or branched alkyl or alkenyl of 1 to 10 carbon atoms, cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic, aromatic or heterocycloalkyl residues, and further where one of the substituents may be H, and where the acyl group has a linear chain length of at least three carbon atoms, between the carbonyl functionality and the sulfur atom. ;;Such additional maytansines include compounds represented by formula (III): ;;;;;wherein: ;;Y’ represents (CR7R8)l(CR9=CR10)p(CΞC)qAr(CR5R6)mDu(CR11=CR12)r(CΞC)sBt(CR3R4)nCR1 R2SZ, ;;wherein: ;;R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic, aromatic or heterocycloalkyl, and in addition R2 may be H; ;;A, B, D are cycloalkyl or cycloalkenyl of 3 to 10 carbon atoms, single or substituted aryl or heterocyclic, aromatic or heterocycloalkyl; ;R3, R4, R5, R6, R7, R8, R9, R11 and R12 are each independently H, CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl; ;;l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any time; and ;;Z is H, SR or -COR, where R is linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic including heterocycloalkyl. ;;Preferred embodiments of formula (III) include compounds of formula (III), wherein: ;;R1 is H, R2 is methyl and Z is H; ;R1 and R2 are methyl and Z is H; ;R1 is H, R2 is methyl and Z is –SCH3; ;R1 and R2 are methyl and Z is –SCH3. ;;Such additional maytansines also include compounds represented by formula (IV-L), (IV-D) or (IV-D,L): ;;;;;;(IV-L) (IV-D) (IV-D,L) ;;wherein: ;;Y is (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ, ;;wherein: ;R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl, and in addition R2 may be H; ;;R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl; ;;l, m and n are each independently an integer from 1 to 5, and in addition n may be 0; ;;Z is H, SR or –COR, where R is linear or branched alkyl or alkenyl of from 1 to 10 carbon atoms, cyclic alkyl or alkenyl of 3 to 10 carbon atoms, or simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl; and ;;May represents a maytansinoid bearing the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl. ;;Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) include compounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein: ;;R 1 is H, R 2 is methyl, R 5 , R 6 , R 7 and R 8 are each H, l and m are each 1, n is 0 and z is H; R1 and R2 are methyl, R5, R6, R7, R8 are each H, l and m are 1, n is 0 and Z is H; ;;R1 is H, R2 is methyl, R5, R6, R7, R8 are each H, l and m are each 1, n is 0 and Z is –SCH3; ;;R1 and R2 are methyl, R5, R6, R7, R8 are each H, l and m are 1, n is 0 and Z is –SCH3. ;;Preferably, the cytotoxic agent is represented by formula (IV-L). ;Such additional maytansines also include compounds represented by formula (V): ;;;;;;wherein: ;;Y is (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ, ;;wherein: ;;R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl, and in addition R2 may be H; ;;R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl; ;;l, m and n are each independently an integer from 1 to 5, and in addition n may be 0; and ;;Z is H, SR or -COR, where R is linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic including heterocycloalkyl. ;;Preferred embodiments of formula (V) include compounds of formula (V), wherein: ;R 1 is H, R 2 is methyl, R 5 , R 6 , R 7 and R 8 are each H; l and m each are 1; n is 0; and Z is H; R1 and R2 are methyl, R5, R6, R7 and R8 are each H, l and m are 1; n is 0; and Z is H; ;;R1 is H, R2 is methyl, R5, R6, R7 and R8 are each H, l and m are each 1, n is 0 and Z is –SCH3; ;;R1 and R2 are methyl, R5, R6, R7 and R8 are each H, l and m are 1, n is 0 and Z is –SCH3. ;;Such additional maytansines further include compounds represented by the formula (VI-L), (VI-D) or (VI-D,L): ;;;;;(VI-L) (VI-D) (VI-D, L) ;;wherein: ;;Y2 is (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ2, ;;wherein: ;;R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and in addition R2 may be H; ;;R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear, cyclic alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl; ;;l, m and n are each independently an integer from 1 to 5, and in addition n can be 0; ;Z2 is SR or COR, where R is a linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl; and ;;May is a maytansinoid. ;;Such additional maytansines also include compounds represented by formula (VII): ;;;;;wherein: ;;Y2’ is (CR7R8)l(CR9=CR10)p(CΞC)qAr(CR5R6)mDu(CR11=CR12)r (CΞC)sBt(CR3R4)nCR1R2SZ2, ;;wherein: ;;R1 and R2 are each independently CH3, C2H5, linear, branched alkyl or alkenyl of 1 to 10 carbon atoms, cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl, and in addition R2 may be H; ;;A, B and D are each independently cycloalkyl or cycloalkenyl of 3 to 10 carbon atoms, simple or substituted aryl, heterocyclic aromatic or heterocycloalkyl; ;R3, R4, R5, R6, R7, R8, R9, R11 and R12 are each independently H, CH3, C2H5, linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl; ;;l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not 0 at the same time; and ;;Z2 is SR or -COR, where R is linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl. ;;Preferred embodiments include compounds of formula (VII) wherein R1 is H and R2 is methyl. ;;The above-mentioned maytansinoids may be conjugated to an anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 or a homolog or fragment thereof, wherein the antibody is linked to the maytansinoid using the thiol or disulfide functionality present on the acyl group of an acylated amino side chain found at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl of the maytansinoid, and wherein the acyl group in the acylated amino acid side chain has its thiol or disulfide functionality located on a carbon atom having one or two substituents, which are CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl, and in addition one of the substituents may be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl functionality and the sulfur atom. ;A preferred conjugate is one comprising the anti-CD38 antibody 38SB19, conjugated to a maytansinoid of formula (VIII): ;;;;;wherein: ;;Y1’ is (CR7R8)l(CR9=CR10)p(CΞC)qAr(CR5R6)mDu(CR11=CR12)r (CΞC)sBt(CR3R4)nCR1R2S-, ;;wherein: ;;A, B and D are each independently cycloalkyl or cycloalkenyl of 3 to 10 carbon atoms, simple or substituted aryl or heterocyclic aromatic, or heterocycloalkyl; ;;R3, R4, R5, R6, R7, R8, R9, R11 and R12 are each independently H, CH3, C2H5, linear alkyl or alkenyl of from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl; and ;;l, m, n, o, p, q, r, s and t are each independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not 0 simultaneously. ;It is preferred that R1 is H and R2 is methyl or R1 and R2 are methyl. ;;A still more preferred conjugate is one comprising anti-CD38 antibody 38SB19 conjugated to a maytansinoid of the formula (IX-L), (IX-D) or (IX-D,L): ;;;;wherein: ;;Y1 is (CR7R8)l(CR5R6)m(CR3R4)nCR1R2S-, ;;wherein: ;;R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of from 3 to 10 carbon atoms, phenyl, substituted phenyl, heterocyclic aromatic or heterocycloalkyl, and in addition R2 may be H; ;;R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or alkenyl of 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl of 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic including heterocycloalkyl; ;;l, m and n are each independently an integer from 1 to 5, and in addition n can be 9; and ;;May represents a maytansinol bearing the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl. ;;Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D,L) include compounds of formulas (IX-L), (IX-D) and (IX-D,L) wherein: ;;R1 is H and R2 is methyl or R1 and R2 are methyl; ;;R1 is H, R2 is methyl, R5, R6, R7 and R8 are each H; l and m are each 0; n is 0; R1 and R2 are methyl; R5, R6, R7 and R8 are each H; l and m are 1; n is 0. ;;Preferably, the cytotoxic agent is represented by formula (IX-L). ;;A further preferred conjugate is one comprising the anti-CD38 antibody 38SB19 conjugated to a maytansinoid of formula (X): ;;;;;wherein the substituents are as defined for formula (IX) above. ;;Especially preferred is any of the above-described compounds, wherein R1 is H, R2 is methyl, R5, R6, R7 and R8 are each H, l and m are each 1 and n is 0. ;;Furthermore especially preferred is any of the above-described compounds, wherein R1 and R2 are methyl, R5, R6, R7, R8 are each H, l and m are 1 and n is 0. ;;Furthermore, the L-aminoacyl stereoisomer is preferred. ;;Any of the maytansinoids described in US application 10/84136 of May 20, 2004, can also be used in the cytotoxic conjugate. ;Disulfide-containing linker groups ;To connect the maytansinoid to a cell binding agent such as the 28SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody, the maytansinoid comprises a linker moiety. This linker moiety contains a chemical bond that allows for the release of fully active maytansinoids at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase-labile bonds and esterase-labile bonds. Disulfide bonds are preferred. ;;The linker moiety also comprises a reactive chemical group. The reactive chemical group may be covalently linked to the maytansinoid via a disulfide bond linker moiety. ;;Especially preferred reactive chemical groups are N-succinimidyl esters and N-sulfosuccinimidyl esters. ;;Especially preferred maytansinoids comprise a linker moiety containing a reactive chemical group and are C-3 esters of maytansinol and its analogs wherein the linker moiety contains a disulfide bond and a chemically reactive group comprising an N-succinimidyl or N-sulfosuccinimidyl ester. ;;Many positions on maytansinoids can serve as positions for chemically linking the linker moiety. For example, the C-3 position is a hydroxyl group, the C-14 position is modified with hydroxymethyl, the C-15 position is modified with hydroxy, and the C-20 position is a hydroxy group, or are expected to be useful. However, the C-3 position is preferred and the C-3 position of maytansinol is particularly preferred. ;;While the synthesis of esters of maytansinol with a linker moiety is described in terms of disulfide bond-containing linker moieties, those skilled in the art will appreciate that linker moieties with other chemical linkages (as described above) may also be utilized in the same manner as other maytansinoids. Specific examples of other chemical linkages include acid-labile linkages, peptidase-labile linkages, and esterase-labile linkages. The disclosure in US 5,208,020 teaches the preparation of maytansinoids having such linkages. ;;The synthesis of maytansinoids and maytansinoid derivatives with a disulfide moiety bearing a reactive group is described in US 6,441,163 and 6,333,410 as well as US application 10/161,651. ;;Maytansinoids containing the reactive group such as DM1 are reacted with an antibody such as 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody to give cytotoxic conjugates. These conjugates can be purified by HPLC or gel filtration. ;Several excellent schemes for preparing such antibody-maytansinoid conjugates are provided in US 6333 410 as well as in US applications 09/867598, 10/161651 and 10/024 290. ;;In general, a solution of an antibody in aqueous buffer can be incubated with a molar excess of maytansinoid having a disulfide moiety bearing a reactive group. ;The reaction mixture can be quenched by the addition of an excess of amine (such as ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration. ;;The number of maytansinoid molecules bound per antibody molecule can be determined by spectrophotometrically measuring the ratio of absorbance at 252 nm to 280 nm. An average of 1-10 maytansinoid molecules per antibody molecule is preferred. ;;Conjugates of antibodies with maytansinoid drugs can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro. For example, cell lines such as the human lymphoma cell line Daudi, the human lymphoma cell line Ramos, the human multiple myeloma cell line MOLP-8, and the human T-acute lymphocytic leukemia line MOLT-4 can readily be used to assess the cytotoxicity of these compounds. Cells for evaluation can be exposed to the compound for 24 hours and the surviving percentages of cells measured in direct assays in a manner known per se. IC50 values can then be calculated from the results of these assays. ;;PEG-Containing Linker Groups ;Maytansinoids can also be linked to cell binding agents using PEG linker groups as disclosed in U.S. Application No. 10/024,290. These PEG linker groups are soluble in both water and non-aqueous solvents, and can be used to link one or more cytotoxic agents to a cell binding agent. Examples of PEG linker groups include heterobifunctional PEG linkers that link to cytotoxic agents and cell binding agents at opposite ends of the linker via a functional sulfhydryl or disulfide group on one end, and an active ester on the other end. ;;As a general example of the synthesis of a cytotoxic conjugate using a PEG linker group, reference is again made to U.S. Application No. 10/024,290 for specific details. The synthesis begins with the reaction of one or more cytotoxic agents bearing a reactive PEG moiety with a cell binding agent, resulting in the displacement of the terminal active ester of each reactive PEG moiety by an amino acid residue from the cell binding agent, thereby providing a cytotoxic conjugate comprising one or more cytotoxic agents covalently linked to a cell binding agent via a PEG linker group. ;;Taxanes ;The cytotoxic agent used in the cytotoxic conjugates of the disclosure may also be a taxane or a derivative thereof. ;;Taxanes are a family of compounds that include paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic derivative, two compounds that are widely used in the treatment of cancer. Taxanes are mitotic spindle poisons that inhibit the depolymerization of tubulin and result in cell death. While docetaxel and paclitaxel are useful agents in the treatment of cancer, their antitumor activity is limited due to their non-specific toxicity against normal cells. Furthermore, compounds such as paclitaxel and docetaxel are not sufficiently potent per se to be used in conjugates of cell binding agents. ;;A preferred taxane for use in the preparation of cytotoxic conjugates is a taxane of formula (XI): ;;;;;;Methods for synthesizing taxanes that can be used in the cytotoxic conjugates, together with methods for conjugating the taxanes to cell binding agents such as antibodies, are described in detail in US 5,416,064, 5,475,092, 6,340,701, 6,372,738 and 6,436,931 as well as in US applications 10/024290, 10/144042, 10/207814, 10/210112 and 10/369,563. ;Tomaymycin Derivatives ;The cytotoxic agents may also comprise a tomaymycin derivative. Tomaymycin derivatives are pyrrolo[1,4]benzodiazepines (PBDs), a known class of compounds that exert their biological properties by covalently binding to the N2 of guanine in the minor groove of DNA. PBDs include a number of minor groove binders such as anthramycin, neotramycin and DC-81. ;;New tomaymycin derivatives that retain high cytotoxicity and can be effectively linked to cell binding agents are described in PCT/IB2007/000142. ;The cell binding agent-tomaymycin derivative complexes allow the full cytotoxic effect of the tomaymycin derivatives to be applied in a targeted manner only to unwanted cells, thereby avoiding side effects due to damage to untargeted, healthy cells. ;;The cytotoxic agent according to the disclosure comprises one or more tomaymycin derivatives, linked to a cell binding agent such as the 38SB19 antibody, via a linker group. This is part of a chemical moiety that is covalently linked to a tomaymycin derivative in a manner known per se. In a preferred embodiment, the chemical moiety may be covalently linked to the tomaymycin derivative via a disulfide bond. ;;The tomaymycin derivatives useful according to the disclosure have formula (XII), as shown below: ;;;;;;where ;;----- represents an optional single bond; ;;----- represents either a single or a double bond; ;provided that when ----- represents a single bond, U and U', the same or different, independently represent H, and W and W', the same or different, independently are selected from the group consisting of OH, an ether such as -OR, an ester (for example an acetate) such as -OCOR, a carbonate such as -OCOOR, a carbamate such as -OCONRR', a cyclic carbamate such as -OCONRR', a urea such as -NRCONRR', a thiocarbamate such as -OCSNHR, a cyclic thiocarbamate such as -SH, a sulfide such as -SR, a sulfoxide such as -SOR, a sulfone such as -SOOR, a sulfonate such as -SO3, a sulfonamide such as -NRSOOR, an amine such as -NRR', optionally a cyclic amine such as N10 and C11 are part of the cycle, a hydroxylamine derivative such as -NROR', an amide such as -NRCOR, an azido such as –N3, a cyano-, a halo-, a trialkyl- or triarylphosphonium-, or an amino acid-derived group; preferably W and W’ are the same or different and are OH, Ome, Oet, NHCONH2, SMe; ;;and when ----- is a double bond, U and U’ are absent and W and W’ represent H; ;;R1, R2, R1’ and R2’ are the same or different and independently are selected from halide or alkyl optionally substituted with one or more Hal, Cn, NRR’, CF3, OR, aryl, Het, S(O)qR, or R1 and R2 and R1’ and R2’ together form a double bond-containing group =B, respectively =B’; ;;Preferably R1 and R2 and R1’ and R2’ together form a double bond-containing group =B, respectively =B’; ;;B and B’ are the same or different and independently selected from alkenyl optionally substituted with one or more Hal, CN, NRR’, CF3, OR, aryl, Het, S(O)qR or B and B’ represent an oxygen atom; ;;Preferably B=B’. ;;More preferably B=B’= =CH2 or =CH-CH3, ;;X, X’ are the same or different and independently selected from one or more of –O-, -NR-, -(C=O)-, -S(O)q-. ;Preferably X=X’. ;;More preferably X=X’=O. ;A, A’ are the same or different and independently selected from alkyl or alkenyl independently containing an oxygen, nitrogen or sulfur atom, each optionally substituted with one or more Hal, CN, NRR’, CF3, OR, S(O)qR, aryl, Het, alkyl, alkenyl. ;;Preferably A=A’. ;;More preferably A=A’=linear, unsubstituted alkyl. ;;Y, Y’ are the same or different and independently selected from H or OR. ;;Preferably Y=Y’. ;;Most preferably Y=Y’=OAlkyl, and especially OMethyl. ;;T is –NR-, -O-, -S(O)q- or a 4- to 10-membered aryl, cycloalkyl, heterocycle or heteroaryl, each optionally substituted with one or more Hal, CN, NRR’, CF3, R, OR, S(O)qR and/or one or more linkers, or a branched alkyl, optionally substituted with one or more Hal, CN, NRR’, CF3, OR, S(O)qR and/or one or more linkers, or a linear alkyl substituted with one or more Hal, CN, NRR’, CF3, OR, S(O)qR and/or one or more linkers. ;;Preferably T is a 4- to 10-membered aryl or heteroaryl, more particularly phenyl or pyridyl, optionally substituted with one or more linkers. ;;Said linker comprises a connecting group. Suitable ones are well known in the art and include thiol, sulfide or disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Disulfide groups and thioether groups are preferred. ;;When the linker group is a thiol-, sulfide- (so-called thioether-S-) or disulfide (-S-S-)-containing group, the side chain carrying the thiol, sulfide or disulfide group may be straight or branched, aromatic or heterocyclic. Suitable side chains can be readily identified by those skilled in the art. ;;Preferably, the linker has the formula: ;;-G-D-(Z)p-S-Z’ ;;where ;;G is a single or double bond, -O-, -S- or –NR-; ;;D is a single bond or –E-, -E-NR-, -E-NR-F-, -E-O-, -E-O-F-, -E-NR-CO-, -E-NR-CO-F-, -E-CO-, -CO-E-, -E-CO-F, -E-S-, -E-S-F-, -E-NR-C-S-, -E-NR-CS-F-; ;;where E and F are the same or different and are independently selected from linear or branched –(OCH2CH2)iAlkyl(OCH2CH2)j-, -Alkyl(OCH2CH2)i-Alkyl-, -(OCH2CH2)i-, -(OCH2CH2)iCycloalkyl(OCH2CH2)j-, -(OCH2CH2)iHeterocycle(OCH2CH2)j-, -(OCH2CH2)iAryl(OCH2CH2)j-, -(OCH2CH2)iHeteroaryl(OCH2CH2)j-, –Alkyl-(OCH2CH2)iAlkyl(OCH2CH2)j-, -Alkyl-(OCH2CH2)i-, -Alkyl-(OCH2CH2)i-Cycloalkyl(OCH2CH2)j-, -Alkyl(OCH2CH2)iHeterocycle(OCH2CH2)j-, -Alkyl-(OCH2CH2)iAryl(OCH2CH2)j-, -Alkyl(OCH2CH2)iHeteroaryl(OCH2CH2)j-, -Cycloalkyl-Alkyl-, -Alkyl-Cycloalkyl-, -Heterocycle-Alkyl-, -Alkyl-Heterocycle-, -Alkyl-Aryl-, -Aryl-Alkyl-, -Alkyl-Heteroaryl-, -Heteroaryl-Alkyl-; ;;wherein i and j, the same or different, are integers and are independently selected from 0, 1 to 2000; ;;Z is straight or branched alkyl; ;;p is 0 or 1; ;;Z’ is H, a thiol protecting group such as COR, R20 or SR20, where R20 is H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocycle, provided that when Z’ is H, the compound is in equilibrium with the corresponding compound formed by intramolecular cyclization resulting from the addition of the thiol group –SH to the imine bond –NH= of one of the PBD moieties; ;n, n’ are the same or different and are 0 or 1; ;;q is 0, 1 or 2; ;;R, R’ are the same or different and are independently selected from H, alkyl, aryl, each optionally substituted with Hal, CN, NRR’, CF3, R, OR, S(O)qR, aryl, Het; ;or their pharmaceutically acceptable salts, hydrates or hydrated salts, or polymorphic, crystalline structures of these compounds, or their optical isomers, racemates, diastereomers or enantiomers. ;;The compounds of the general formula (XII) with geometric and stereoisomers are also part of the description. ;;The N.10, C-11 double bond in the tomaymycin derivatives of formula (XII) is known to be readily convertible in a reversible manner to the corresponding imine adducts in the presence of water, an alcohol, a thiol, a primary or secondary amine, urea and other nucleophiles. This process is reversible and can readily regenerate the corresponding tomaymycin derivative in the presence of a dehydrating agent in a non-protic organic solvent, under vacuum or at elevated temperatures (Z. Tozuka, 1983, J. Antibiotics, 36: 276). ;;Thus, reversible derivatives of tomaymycin derivatives of the general formula (XIII) can also be used: ;;;;;;where A, X, Y, n T, A', X', Y', n', R1, R2, R1', R2' are defined as under formula (XII) and W and W' are the same or different and are selected from the group consisting of OH, an ether such as -OR, an ester (for example an acetate) such as -OCOR, -COOR, a carbonate such as -OCOOR, a carbamate such as -OCONRR', a cyclic carbamate such as N10 and C11 are part of the ring, a urea such as -NRCONRR', a thiocarbamate such as -OCSNHR, a cyclic thiocarbamate such as N10 and C11 are part of the ring, -SH, a sulfide such as -SR, a sulfoxide such as -SOR, a sulfone such as -SOOR, a sulfonate such as -SO3-, a sulfonamide such as –NRSOOR, an amine such as –NRR’, optionally a cyclic amine such that N10 and C11 are part of the cycle, a hydroxylamine derivative such as –NROR’, an amide such as –NRCOR, -NRCONRR’, an azido such as –NR, a cyano, halo, trialkyl or triarylphosphonium, an amino acid derived group. Preferably W and W’ are the same or different and are OH, Ome, Oet, NHCONH2, SMe. ;;Compounds of formula (XIII) may thus be considered as solvates which include water when the solvent is water, these solvates may be particularly useful. ;In a preferred embodiment, the tomaymycin derivatives according to the invention are selected from the group comprising: ;;8,8’-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]; ;;8,8’-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]; ;;8,8'-[1,5-pentanediylbis(oxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[1,4-butanediylbis(oxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyleneoxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepine-5-one]; ;;8,8'-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; 8,8'-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(methyleneoxy)]-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-{5-[3-(4-methyl-4-methyldisulfanylpentanoylamino)propyloxy]-1,3-benzenediylbis-(methyleneoxy)}-bis[(S)-2-et-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepine-8-yloxy]-ethyl}-carbamic acid tert-butyl ester; ;;8,8'-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(4-(2-(4-mercapto-4-methyl)-pentanamidoethoxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;8,8'-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamidoethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]-benzodiazepine-5-one]; ;;8,8'-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamidopropoxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4] benzodiazepine-5-one]; ;8,8'-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamidobutoxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]-benzodiazepine-5-one]; ;;8,8'-[(4-(3-[4-(4-methyl-4-methyldisulfanylpentanoyl)-piperazin-1-yl]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(1-(3-[4-(4-methyl-4-methyldisulfanylpentanoyl)-piperazin-1-yl]-propyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanylpentanoylamino)-ethoxy]-ethoxy}-ethoxy)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanylpentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-be nzen-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanylpentanoylamino)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanylpentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-py ridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; 8,8'-[(1-(2-[methyl-(2-methyl-2-methyldisulfanylpropyl)-amino]-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8'-[(4-(3-[methyl-(4-methyl-4-methyldisulfanylpentanoyl)-amino]-propyl)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;8,8'-[(4-(3-[methyl-(2-methyl-2-methyldisulfanylpropyl)-amino]-propyl)-pyridine-2,6-dimethyl)-dioxy]-bis[(S)-2-et-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one]; ;;8,8’-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-one] ;;as well as the corresponding mercapto derivatives, or their pharmaceutically acceptable salts, hydrates or hydrated salts, or the polymorphic, crystalline structures of these compounds, or their optical isomers, racemates, diastereomers or enantiomers. ;;Preferred compounds are those of the formula: ;;;;or ;;;;;wherein X, X’, A, A’, Y, Y’ ̈, T, n, n’ are as defined above. ;;The compounds of formula (XII) can be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by using or adapting methods as described below, or variations thereof as will be known to the skilled person. The appropriate modifications and substitutions will be readily apparent and well known or can be readily obtained from the scientific literature, to those skilled in the art. In particular, such methods can be found in R.C. Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999. ;;Methods for synthesizing tomaymycin derivatives that can be used are described in PCT/IB2007/000142. Compounds according to the disclosure can be prepared by a number of synthetic routes. The reagents and starting materials are commercially available, or can be readily synthesized by techniques known per se to those skilled in the art (see, for example, WO 00/12508, WO 00/12507, WO 2005/040170, WO 2005/085260, FR1516743, M. Mori et al., 1986, Tetrahedron, 42: 3793-3806). ;;The conjugate molecules of the disclosure can be formed using any technique. The tomaymycin derivatives can be linked to an antibody or other cell-binding agent via an acid-labile linker, or via a photolabile linker. The derivatives can be condensed with a peptide of a suitable sequence and then coupled to a cell-binding agent to provide a peptidase-labile linker. The conjugates can be prepared to contain a primary hydroxyl group that can be succinylated and linked to a cell binding agent to give a conjugate that can be cleaved by intracellular esterases to release the free derivative. ;Preferably, the derivatives are synthesized to contain a free or protected thiol group, and then one or more disulfide or thiol-containing derivatives are each covalently linked to the cell binding agent via a disulfide bond or a thioether link. ;Numerous methods of conjugation are described in US 5,416,064 and 5,475,092. ;The tomaymycin derivatives can be modified to give a free amino group and then linked to an antibody or other cell binding agent via an acid-labile linker or a photolabile linker. The tomaymycin derivatives with a free amino or carboxyl group can be condensed with a peptide and then linked to a cell binding agent to give a peptidase-labile linker. The tomaymycin derivatives with a free hydroxyl group on the linker can be succinylated and linked to a cell binding agent to give a conjugate that can be cleaved by intracellular esterases to release free drug. Preferably, the tomaymycin derivatives are treated to form a free or protected thiol group, after which the disulfide or thiol-containing tomaymycin dimers are linked to the cell binding agent via disulfide bonds. ;Preferably, monoclonal antibody or cell binding agent-tomaymycin derivative conjugates are those linked via a disulfide bond as described above, which are capable of delivering tomaymycin derivatives. Such cell binding conjugates are prepared in a manner known per se such as by modification of monoclonal antibodies with succinimidylpyridyl dithiopropionate (SPDP) (Carlsson et al., 1978, Biochem. J., 173: 723-737). The resulting thiopyridyl group is then displaced by treatment with thiol-containing tomaymycin derivatives to give disulfide-linked conjugates. Alternatively, and in the case of aryldithio-tomaymycin derivatives, the formation of the cell-binding conjugate is accomplished by direct displacement of the aryl-thiol in the tomaymycin derivative with sulfhydryl groups previously introduced into antibody molecules. Conjugates containing 1 to 10 tomaymycin derivatives linked via a disulfide bridge can be readily prepared by these methods. ;;More specifically, a solution of the dithio-nitropyridyl-modified antibody at a concentration of 2.5 mg/ml in 0.05 M potassium phosphate buffer, at pH 7.5 containing 2 mM EDTA, is treated with the thiol-containing tomaymycin derivative (1.3 molar equivalents per dithiopyridyl group). The release of the thio-nitropyridine from the modified antibody is monitored spectrophotometrically at 325 nm and is complete within about 16 hours. The antibody-tomaymycin derivative conjugate is purified and freed from unreacted drug and other low molecular weight material by gel filtration through a column of Sephadex G-25 or Sephacryl S300. The number of tomaymycin derivative moieties bound per antibody molecule can be determined by measuring the ratio of the absorbance at 230 nm to 275 nm. An average of 1-10 tomaymycin derivative molecules per antibody molecule can be linked via disulfide bonds by this method. ;;The effect of conjugation on the binding affinity to the antigen-expressing cells can be determined using the methods previously described by Liu et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 8618-8623. The cytotoxicity of the tomaymycin derivatives and their antibody conjugates against cell lines can be measured by back-extrapolation of cell proliferation curves as described in Goldmacher et al., 1985, J. Immunol., 135: 3648-3651. The cytotoxicity of these compounds to adherent cell lines can be determined by clonogenic assays as described in Goldmacher et al., 1986, J. Cell Biol., 102: 1312-1319. ;;Leptomycin Derivatives ;The cytotoxic agent of the disclosure may also comprise a leptomycin derivative. ;"Leptomycin derivatives" refers to members of the leptomycin family as defined in Kalesse et al. (2002, Synthesis 8: 981-1003) and include: leptomycins such as laptomycin A and leptomycin B, callystatins, ratjadones such as ratjadone A and ratjadone B, anguinomycins such as anguinomycin A, B, C, D, kasumycins, leptolstatin, leptofuranins such as leptofuranin A, B, C, D. Derivatives of leptomycin A and B are preferred. ;;More particularly, the derivatives have formula (I): ;;;;wherein ;;Ra and Ra' are H or -Alk; it is preferred that Ra is -Alk, and especially methyl and Ra' is H; ;;R17 is alkyl optionally substituted with OR, CN, NRR' or perfluoroalkyl; preferably R17 is alkyl, and more especially methyl or ethyl; ;;R9 is alkyl optionally substituted with OR, CN, NRR', perfluoroalkyl; preferably R9 is alkyl, and more particularly methyl; ;;X is –O- or –NR, and it is preferred that an X is –NR-; ;;Y is –U-, -NR-U-, -O-U-, -NR-CO-U-, -U-NR-CO-, -U-CO-, -CO-U- ; ;preferably, when X is –O-, Y is –U-, -NR-U-, -U-NR-CO-; ;;wherein U is selected from straight or branched –Alk-, -Alk(OCH2CH2)m-, -(OCH2CH2)m-Alk-, -Alk(OCH2CH2)m-Alk-, -(OCH2CH2)m-, -Cycloalkyl-, -Heterocycle-, -Cycloalkyl-Alk-, -Alk-Cycloalkyl-, -Heterocycle-Alk-, -Alk-Heterocycle-; ;;wherein m is an integer selected from 1 to 2000; ;preferably U is linear or branched –Alk-; ;;Z is –Alk-; ;;n is 0 or 1, and preferably n is 0; ;;T is H, a thiol protecting group such as Ac, R1 or SR1, where R1 is H, methyl, Alk, cycloalkyl, optionally substituted aryl or heterocycle, or T represents ;;;;;wherein: ;;Ra, Ra’, R17, R9, X, Y, Z and n are as defined above; ;;preferably T is H or SR1, where R1 is Alk, and more particularly methyl; ;;R and R’ are the same or different and are H or alkyl; ;;Alk is straight or branched alkyl, preferably Alk represents (-(CH2-q(CH2)q)p-, where p is an integer from 1 to 10; and q is an integer from 0 to 2; preferably Alk represents –(CH2)- or –C(CH3)2-; ;;or their pharmaceutically acceptable salts, hydrates or hydrated salts, or polymorphic, crystalline structures of these compounds, or their optical isomers, racemates, diastereomers or enantiomers. ;;Preferred compounds can be selected from: ;;(2-Methylsulfanylethyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5, 7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid; ;Bis-[(2-mercaptoethyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5, 7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid]; ;;(2-Mercaptoethyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid; ;;(2-Methyldisulfanylethyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5, 7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid; ;;(2-Methyl-2-methyldisulfanylpropyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid; ;;(2-Mercapto-2-methylpropyl)-amide of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic acid ;;or their pharmaceutically acceptable salts, hydrates or hydrated salts, or the polymorphic, crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers. ;;To link derivatives to a cell binding agent, the derivative must include a moiety (a linker group) that allows the derivative to be linked to a cell binding agent via a linker such as a disulfide bond, a sulfide (or thioether bond included therein), an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group. The derivatives are prepared such that they contain a moiety necessary to link leptomycin derivatives to a cell binding agent via, for example, a disulfide bond, a thioether bond, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group. To further increase solubility in aqueous solutions, the linker group may contain a polyethylene glycol spacer. Preferably, a sulfide or disulfide linker is used because the reducing environment of the target cell results in cleavage of the sulfide or disulfide, releasing the derivatives with an associated increase in cytotoxicity. ;Compounds of the disclosure may be prepared via a number of synthetic routes. Reagents and starting materials are commercially available, or readily synthesizable via techniques well known to those skilled in the art. Methods for synthesizing leptomycin derivatives that can be used in the cytotoxic conjugates, together with methods for conjugating said leptomycin derivatives to cell binding agents such as antibodies, are described in detail in EP application 06290948.6. ;;CC-1065 analogs ;The cytotoxic agent used in the cytotoxic conjugates according to the disclosure may also be CC-1065 or a derivative thereof. ;;CC-1065 is a potent antitumor antibiotic isolated from the culture broth of Streptomyces zelensis. CC-1065 is about 1000 times more potent in vitro than commonly used anti-cancer agents such as doxorubicin, methotrexate and vincristine (B.K. Bhuyan et al., 1982, Cancer Res., 42, 3532-3537). CC-1065 and its analogs are described in US 6372 738, 6340 701, 5846 545 and 5585 499. ;;The cytotoxic potency of CC-1065 is correlated with its alkylating activity and its DNA binding or DNA intercalating activity. These two activities reside in separate parts of the molecule. Thus, the alkylating activity is found in the cyclopropapyrroloindole (CPI) subunit, and the DNA binding activity resides in the two pyrroloindole subunits. ;;Although CC-1065 has certain attractive features as a cytotoxic agent, the compound has limitations in therapeutic use. Administration of CC-1065 to mice causes delayed hepatotoxicity leading to mortality on day 50 after a single intravenous dose of 12.5 µg/kg (V. L. Reynolds et al., 1986, J. Antibiotics, XXIX: 319-334). This has prompted attempts to develop analogues that do not cause delayed toxicity, and the synthesis of simpler analogues modeled on CC-1065 has been described (M.A. Warpehoski et al., 1988, J. Med. Chem., 31: 590-603). ;;In another series of analogs, the CPI moiety was replaced by a cyclopropabenzindole (CBI) moiety (D.L. Boger et al., 1990, J. Org. Chem., 55: 5823-5833; D.L. Boger et al., 1991, BioOrg. Med. Chem. Lett., 1: 115-120). These compounds maintain the high in vitro potency of the parent drug without causing delayed toxicity in mice. As in CC-1065, these compounds are alkylating agents that bind to the minor groove of DNA in a covalent manner and cause cell death. However, clinical evaluation of the most promising analogs, adozelesin and carzelesin, has led to disappointing results (B.F. Foster et al., 1996, Investigational New Drugs, 13: 321-326; I. Wolff et al., 1996, Clin. Cancer Res., 2: 1717-1723). These drugs show poor therapeutic effects due to the high systemic toxicity. ;;The therapeutic efficacy of CC-1065 analogs can be greatly improved by altering the in vivo distribution via targeted delivery to the tumor site, resulting in lower toxicity to non-target tissues, and thereby lower systemic toxicity. To achieve this goal, conjugates of analogs and derivatives of CC-1065 with cell-binding agents that specifically target tumor cells have been described (US 5,475,092, 5,585,499, 5,846,545). These conjugates typically exhibit high target-specific cytotoxicity in vitro, and exceptional anti-tumor activity in human tumor xenograft models in mice (R.V. J. Chari et al., 1995, Cancer Res., 55: 4079-4084). ;;Recently, prodrugs of CC-1065 analogs with improved solubility in aqueous media have been described (EP 06290379.4). In these prodrugs, the phenolic group of the alkylating moiety of the molecule is protected with a functionality that makes the drug stable upon storage in acidic aqueous solution, and provides increased aqueous solubility of the drug compared to an unprotected analog. The protecting group can be readily cleaved off in vivo at physiological pH to yield the corresponding active drug. In the prodrugs described in EP 06290379.4, the phenolic substituent is protected as a sulfonic acid-containing phenylcarbamate which has a charge at physiological pH, and therefore has improved water solubility. To further improve water solubility, an optional polyethylene glycol spacer can be introduced into the linker between the indolyl subunit and the cleavable bond as a disulfide group. The introduction of this spacer does not alter the potency of the agent. ;;Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates, along with methods for conjugating analogs to cell binding agents such as antibodies, are described in detail in EP 06290379.4, in US 5475 092, 5846 545, 5 585 499, 6534 660 and 6586 618 and in US applications 10/116053 and 10/265452. ;Other Drugs ;Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs, dolastatin and dolastatin analogs are also suitable for preparing conjugates. The drug molecules may also be linked to the antibody molecules via an intermediate carrier molecule such as serum albumin. ;Doxarubicin and danorubicin compounds, such as those described in US SN 09/740991, may also be useful cytotoxic agents. ;;Therapeutic Compositions ;Also described is a therapeutic composition for treating a hyperproliferative disorder, or inflammatory disease, or an autoimmune disease in a mammal, comprising a therapeutically effective amount of a compound of the disclosure, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is for treating cancer including (but not limited to) B-cell lymphoma, and multiple myeloma. ;The present disclosure describes pharmaceutical compositions comprising: ;;a) an effective amount of an antibody or an antibody conjugate, and ;;b) a pharmaceutically acceptable carrier which may be inert or physiologically active. ;;As used herein, “pharmaceutically acceptable carriers” include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents, and/or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents such as sugars, polyalcohols, or sodium chlorides, in compositions. Particularly relevant examples of suitable carriers include: (1) Dulbecco’s phosphate buffered saline, pH ~7.4, optionally containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride)) and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20. ;The compositions described herein may also contain an additional therapeutic agent, if necessary for the particular disorder being treated. Preferably, the antibody or antibody conjugate of the disclosure and the supplementary active compound will have complementary activities that do not adversely affect each other. In a preferred embodiment, the additional therapeutic agent is an antagonist of epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF), or an antagonist of a receptor for epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF), including HER2 receptor, HER3 receptor, c-MET and other receptor tyrosine kinases. In a preferred embodiment, the additional therapeutic agent is an agent that targets clusters of differentiation (CD) antigens, including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic or immunomodulatory agent. ;;The compositions may be in a number of forms. These include, for example, liquid, semi-solid or solid dosage forms, but the preferred form depends on the intended route of administration and therapeutic applications. Typically, preferred compositions are in the form of injectable or infusible solutions. The preferred route of administration is parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous) administration. In a preferred embodiment, the compositions are administered intravenously as a bolus or by continuous infusion over a period of time. In another preferred embodiment, they are injected via intramuscular, subcutaneous, intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional routes to exert local as well as systemic therapeutic effects. ;;Sterile compositions for parenteral administration can be prepared by incorporating the antibody or antibody conjugate, in the required amount of the appropriate solvent, followed by sterilization by microfiltration. As the solvent or vehicle, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof, can be used. In many cases, it will be preferable to include isotonic agents such as sugars, polyalcohols, or sodium chloride, in the composition. These preparations may also contain adjuvants and in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile preparations for parenteral administration may also be prepared in the form of sterile solid preparations which may be dissolved at the time of use in sterile water, or any other sterile injectable medium. ;;The antibody or antibody conjugate may also be administered orally. As solid preparations for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules may be used. In these preparations, the active ingredient is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica, under a stream of argon. These preparations may also comprise substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, a colour, a coating (sugar-coated tablet) or a glaze. ;;As liquid preparations for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil may be used. These preparations may include substances other than diluents, for example wetting, sweetening, diluting, flavouring or stabilising products. ;;Doses depend on the desired effect, the duration of treatment and the form of administration used, the dose generally being between 5 mg to 1000 mg per day orally for an adult human with unit doses in the range of 1 mg to 250 mg of active ingredient. In general, the physician will determine the appropriate dosage form depending on the age, weight and other factors specific to the individual being treated.;;Therapeutic Uses ;Described herein is a method of killing a CD38 <+> cell by administering to a patient in need thereof an antibody that binds said CD38 and is capable of killing said CD38 <+ >by apoptosis, ADCC and/or CDC. Any type of antibody or cytotoxic conjugate may be used therapeutically. ;;In preferred embodiments, antibodies or cytotoxic conjugates are used for the treatment of a hyperproliferative disorder or inflammatory disease or autoimmune disease in a mammal. In a more preferred embodiment, one of the pharmaceutical compositions described above, which contains an antibody or cytotoxic conjugate, is used for the treatment of a hyperproliferative disorder in a mammal. In one embodiment, the disorder is a cancer. In particular, the cancer is a metastatic cancer. ;;Accordingly, the pharmaceutical compositions are useful in the treatment or prevention of a number of cancers including, but not limited to, B-cell lymphoma and multiple myeloma. Other diseases include the following: carcinoma, including of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; other tumors including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma and other cancers to be determined where CD38 is expressed. Other disorders include NHL, BL, MED MER, B-CLL, ALL, TCL, AML, HCL, HL or CML, in which CD38 is expressed, and other cancers that can be identified in which CD38 is predominantly expressed. Autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulonephritis, Sjögren's syndrome, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, neuromyelitis optica, Graves' dystrophy, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome and asthma. Other disorders include, for example, rejection reactions such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft-versus-host disease; viral infections such as MV infection, HIV infection, AIDS, etc.; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and others as will be appreciated by those skilled in the art. ;Similarly, the present disclosure provides a method of inhibiting the growth of selected cell populations comprising contacting target cells, or tissues containing the target cells, with an effective amount of an antibody or antibody conjugate, or an antibody or therapeutic agent comprising a cytotoxic conjugate, either alone or in combination with other cytotoxic or therapeutic agents. In a preferred embodiment, the additional therapeutic agent is an antagonist of epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF), or an antagonist of a receptor for epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C, protein S, platelet-derived growth factor (PDGF), heregulin, macrophage stimulating protein (MSP) or vascular endothelial growth factor (VEGF), including HER2 receptor, HER3 receptor, c-MET and other receptor tyrosine kinases. In a preferred embodiment, the additional therapeutic agent is an agent that targets clusters of differentiation (CD) antigens, including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138 and CD152. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic or immunomodulatory agent. ;;The method of inhibiting selected cell populations can be carried out in vitro, in vivo or ex vivo. As used herein, “inhibiting growth” means slowing the growth of a cell, reducing cell viability, causing cell death, lysing a cell and inducing cell death, whether over a short or long period of time. ;;Examples of in vitro uses include treatments of autologous bone marrow prior to transplantation into the same patient to kill diseased or malignant cells; treatments of bone marrow prior to transplantation to kill competent T cells and prevent graft-versus-host disease (GVHD); treatments of cell cultures to kill all cells except the desired variants that do not express the target antigen; or to kill variants that express undesired antigen. ;;The conditions for non-clinical in vitro use can be readily determined by one skilled in the art. ;;Examples of clinical ex vivo use are to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in the treatment of autoimmune disease, or to remove T cells and/or lymphoid cells from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease (GVHD). The treatment can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which a cytotoxic agent has been added. The concentration range is from about 10 µM to 1 pM, for about 30 minutes to about 48 hours, and at about 37°C. The exact conditions of concentration and incubation time, i.e. dose, can be readily determined by one skilled in the art. After incubation, the bone marrow cells are washed with medium containing serum and returned to the patient by intravenous infusion according to known methods. In circumstances where the patient receives other treatment such as ablative chemotherapy or total body irradiation between the time of harvesting the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment. ;;For clinical in vivo use, the antibody, or cytotoxic conjugate, will be supplied as solutions that have been tested for sterility and for endotoxin levels. Examples of suitable protocols for cytotoxic conjugate administration are as follows. Conjugates are administered weekly for 4 weeks as an i.v. bolus every week. Bolus doses are administered in 50 to 100 ml of normal saline, to which 5 to 10 ml of human serum albumin may be added. Doses will be 10 µg to 100 mg per administration i.v. (range 10 ng to 1 mg/kg/day). More preferably, the dose range will be from about 50 µg to 30 mg. Most preferably, the dose range will be from 1 to 20 mg. After four weeks of treatment, the patient may continue to receive treatment on a weekly basis. Specific clinical protocols with respect to route of administration, excipients, diluents, doses, time periods, etc., can be determined by one of ordinary skill in the art, depending on the clinical situation at hand. ;;Diagnostics ;The antibodies described may also be used to detect CD38 in a biological sample in vitro or in vivo. Preferably, the described anti-CD38 antibodies are used in vitro to determine the level of CD38 in a tissue or in cells derived from the tissue. Preferably, the tissue is a diseased tissue. More preferably, the tissue is a tumor or a biopsy thereof. More preferably, the tissue or biopsy thereof is first excised from a patient, and the level of CD38 in the tissue or biopsy can then be determined in an immunoassay with antibodies described herein. The description further provides for the determination of the level of CD38 in a sample of a tissue or biopsy thereof which may be frozen or fixed. The same method can be used to determine other properties of CD38 protein such as cell surface levels, or its cellular location. ;;The above-described method can be used to diagnose a cancer in an individual known to have or suspected of having a cancer, wherein the level of CD38 measured in the patient is compared to that of a normal reference individual or standard. The method can then be used to determine whether a tumor expresses CD38, which may indicate that the tumor will respond well to treatment by the antibodies, antibody fragments or antibody conjugates. Preferably, the tumor is an NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL or CML, in which CD38 is expressed, and other cancers to be determined later in which CD38 is predominantly expressed. ;;The present disclosure further provides monoclonal antibodies, humanized antibodies and epitope-binding fragments thereof that are further labeled for use in research or diagnostic purposes. In preferred embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion. ;;A method of diagnosis is also described wherein said labeled antibodies or epitope-binding fragments thereof are administered to an individual suspected of having a cancer or an inflammatory disease or an autoimmune disease, and the distribution of the label in the body of the individual is measured or followed. ;;Kits ;The present description also includes kits which, for example, comprise a described cytotoxic conjugate and instructions for using the cytotoxic conjugate to kill particular cell types. The instructions may include guidelines for using the cytotoxic conjugates in vitro, in vivo or ex vivo. ;;Typically, the kit will have a compartment containing the cytotoxic conjugate. This may be in lyophilized form, liquid form or any other form that may be included in a kit. Kits may also contain additional elements as necessary, described in the instructions in the kit, for example a sterilized solution for reconstituting the lyophilized powder, additional means for combining with the cytotoxic conjugate prior to administration to a patient, and tools to support administration of the conjugate to a patient. ;Examples ;The invention will now be described with reference to the following examples which are illustrative only and are not intended to limit the invention. ;;Example 1 ;Mouse CD38 antibodies ;300-19 cells, a pre-B cell line derived from Balb/c mice (M. G. Reth et al.1985, Nature, 317: 353-355) which stably express a high level of human CD38 were used for immunization of Balb/c VAF mice. The mice were immunized subcutaneously with about 5 x 10 <6 >CD38-expressing 300-19 cells per mouse every two to three weeks using standard immunization protocols used by ImmunoGen, Inc. The immunized mice were boosted with an additional dose of antigen three days before they were sacrificed for hybridoma generation. The spleens from the mice were collected according to standard animal protocols and ground between two sterile, frosted microscopic plants to obtain a single cell suspension in RPMI-1640 medium. The spleen cells were pelleted, washed, and fused with murine myeloma P3X63Ag8.653 cells (J. F. Kearney et al. 1979, J Immunol, 123: 1548-1550) using polyethylene glycol-1500 (Roche 783641). The fused cells were resuspended in RPMI-1640 selection medium containing hypoxanthine-thymidine (HAT) (Sigma H-2062) and selected for growth in 96-well flat-bottomed culture plates (Corning-Costar 3596, 200 µl of cell suspension per well) at 37ºC (5% CO2). After 5 days of incubation, 100 µl of culture supernatant was removed from each well and replaced with 100 µl of RPMI-1640 medium containing hypoxanthine-thymidine (HT) supplement (Sigma H-0137). Incubation at 37ºC (5% CO2) was continued until hybridoma clones were ready for antibody screening. Other techniques for immunization and hybridoma production may also be used, including those described in J. Langone and H. Vunakis (eds., Methods in Enzymology, Vol.121, “Immunochemical Techniques, Part I”; Academic Press, Florida) and E. Harlow and D. Lane (“Antibodies: A Laboratory Manual”; 1988; Cold Spring Harbor Laboratory Press, New York). ;;By fluorescence-activated cell sorting (FACS) using a Becton Dickinson FACSCalibur or a FACSArray machine, hybridoma culture supernatants were screened (with FITC or PE-conjugated anti-mouse IgG antiserum) for secretion of mouse monoclonal antibodies that bind to the CD38-expressing 300-19 cells but not to the parental 300-19 cells. The hybridoma clones that tested positive were subcloned and the isotype of each secreted anti-CD38 antibody was identified using commercial isotyping reagents (Roche 1493027). A total of 29 antibodies that were positive for CD38 binding were purified by Protein A or –G chromatography using a standard protocol and then characterized further. ;;Example 2 ;Binding characterization of anti-CD38 antibodies ;FACS histograms showing binding of anti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 to CD38-expressing 300-19 cells, and absence of binding to the parental 300-19 cells are shown in Fig. 1. 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 or 38SB39 antibody (10 nM) was incubated for 3 hours with either CD38-expressing 300-19 cells or the parental 300-19 cells (1-2 x 10 <5 >cells per sample) in 100 µl of ice-cold RPMI-1640 medium supplemented with 2% normal goat serum. ;The cells were then pelleted, washed and incubated for 1 hour on ice with FITC-conjugated goat anti-mouse IgG antibody (Jackson Laboratory, 100 µl, 6 µg/ml in cold RPMI-1640 medium supplemented with 2% normal goat serum). The cells were pelleted again, washed, resuspended in 200 µl of PBS containing 1% formaldehyde and analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). ;;FACS histograms of CD38-expressing 300-19 cells incubated with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 showed a strong fluorescence shift compared to that of the negative control (cells incubated with FITC-conjugated goat anti-mouse IgG antibody only) (Fig. 1). Also, no significant fluorescence shift was detected when parental 300-19 cells were incubated with any of these antibodies. Similar results were obtained when the positive control anti-CD38 antibody AT13/5 (Serotec, MCA1019) was used. ;;A strong fluorescence shift was also observed when Ramos (ATCC CRL 1596) lymphoma cells were incubated with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 (Fig. 1). The apparent dissociation constant (K) values for 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 for binding to Ramos cells were estimated from the FACS analysis curves shown in Fig. 2 using the nonlinear regression method for sigmoid dose-response curves (GraphPad Prizm, version 4, program, San Diego, CA). The values are as follows: 0.10 nM, 0.10 nM, 0.12 nM, 0.16 nM, 0.11 nM and 3.3 nM, respectively. ;Example 3 ;Induction of apoptosis of Ramos and Daudi lymphoma cells with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 antibodies ;The anti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 induced apoptosis of Ramos and Daudi (ATCC CCL-213) lymphoma cell lines and the MOLP-8 multiple myeloma cell line (DSMZ ACC 569). The extent of apoptosis was measured by FACS analysis after staining with FITC conjugates of Annexin V (Biosource PHN1018) and with TÆO-PRO-3 (Invitrogen T3605). Annexin V binds phosphotidylserine on the outside, but not on the inside, of the cell membrane bilayer of intact cells. In healthy, normal cells, phosphatidylserine is expressed on the inside of the membrane bilayer, and the transfer of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is one of the earliest detectable signals of apoptosis. Thus, the binding of Annexin V is a signal for the induction of apoptosis. TO-PRO-3 is a monomeric cyanine nucleic acid dye that can only penetrate the plasma membrane when membrane integrity is compromised, as occurs in the later stages of apoptosis. ;;Exceptionally growing cells were plated on plates with approximately 2 x 10 <5 >cells/ml in 24-well plates in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 50 µg/ml gentamycin (referred to below as complete RMPI-1640 medium). Cells were generally grown in complete RMPI-1640 medium unless otherwise stated. Cells were incubated with anti-CD38 antibodies (10 nM) for 24 hours at 37ºC in a humidified atmosphere containing 5% CO2. Cells were then pelleted, washed twice with 500 µl PBS, resuspended in 100 µl binding buffer (provided in the Annexin V-FITC kit) containing 5 µl Annexin V-FITC, and incubated for 15 minutes on ice. Then, 400 µl of binding buffer and TO-PRO-3 (to a final concentration of 1 µM) were added to the mixture and the cell-associated fluorescence of FITC and TO-PRO-3 was immediately measured by FACS. 4000 events were collected for each sample. Dot plots of the fluorescence of TO-PRO-3 (FL4-H; y-axis) and fluorescence of Annexin V-FITC (FL1-H, x-axis) were generated using CellQuest software. ;;The results are shown in Figures 3 and 4. Figure 3 provides an example of such a dot plot for Daudi cells after 24-hour incubation with different anti-CD38 antibodies. The mean percentages of Annexin V-positive cells (including both TO-PRO-3-positive and -negative cells) from duplicate samples were determined from these plots and are shown in Figure 4. Unexpectedly, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 showed strong apoptotic activity. More than 30% of Daudi cells exposed to any of these antibodies were Annexin V-positive compared to only 6% of untreated cells (Figs. 3 and 4A). 38SB13,38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 showed at least 2.4-fold stronger apoptotic activities (24% after subtraction of the untreated control value) than previously known murine CD38 antibodies tested at the same concentration of 10 nM, (AT13/5, OKT10, IB4 and SUN-4B7, less than 10% Annexin V positive after subtraction of the untreated control value) and two other anti-CD38 antibodies generated in the applicant's laboratory (38SB7 and 38SB23, no higher than untreated control, i.e. around 6% Annexin V positive) (Fig. 4A). ;AT13/5 was acquired from Serotec (MCA1019) and OKT10 was produced and purified from hybridoma (ATCC CRL-8022). IB4 and SUN-4B7 were a gift from Prof. F. Malavasi, University of Turin, Italy. Similarly, the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 anti-CD38 antibodies showed at least 3.5-fold stronger pro-apoptotic activity on another lymphoma cell line, Ramos (7% or more Annexin V positive after subtraction of the untreated control value) than any of the previously known murine CD38 antibodies, AT13/5, OKT10, IB4 and SUYN-4B7, or two other novel anti-CD38 antibodies, 38SB7 and 38SB23 (less than 2% Annexin V positive after subtraction of the untreated control value) (Fig. 4B). Finally, the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 anti-CD38 antibodies showed strong pro-apoptotic activity on the multiple myeloma cell line MOLP-8 (Fig. 4C). Approximately 50% of MOLP-8 cells treated with these antibodies were Annexin V positive compared to approximately 39% of untreated cells. In contrast, treatment with the known murine CD38 antibodies, AT13/5, OKT10, IB4 and SUN-4B7, or the two other novel anti-CD38 antibodies, 38SB7 and 38SB23, did not increase the proportion of apoptotic cells. ;;Example 4 ;Cloning and sequencing of light and heavy chains of anti-CD38 antibodies ;;38SB19 antibody ;RNA preparation from hybridoma cells producing 38SB19 antibody. ;;Preparation of total RNA was obtained from 5 x 10 <6>hybrodim cells producing 38SB19 antibody using Qiagen's RNeasy miniprep kit. Briefly, 5 x 10 <6 >cells were pelleted and resuspended in 350 µl RLT buffer (containing 1% β-mercaptoethanol). The suspension was homogenized by passing it through a 21.5 gauge needle and injecting about 10-20 times until it was no longer viscous. Ethanol (350 µl 70% aqueous ethanol) was added to the homogenate and mixed well. The solution was transferred to a spin column, placed in a 2 ml collection tube and spun at >8000 x g for 15 seconds. The column was washed twice with 500 µl RPE buffer, then transferred to a fresh tube and eluted with 30 µl RNase-free water and a 1-minute spin. The 30 µl eluate was placed back on the column for a second 1-minute elution spin. An aliquot of 30 µl of eluant was diluted with water and used to measure UV absorbances at 260 nm for RNA quantitation. ;;cDNA preparation by reverse transcriptase (RT) reaction ;The variable region 38SB19 antibody cDNA was generated from total RNA using Invitrogen's SuperscritII kit. The kit protocol was followed strictly using up to 5 µg of total RNA from Qianeasy mini-preps. Briefly, RNA, 1 µl of working primers and 1 µl of dNTP mix were brought up to 12 µl with RNase-free, sterile, distilled water and incubated at 65ºC for 5 minutes. The mixture was then placed on ice for at least 1 minute. Then 4 µl of 5x reaction buffer, 2 µl of 0.1 M DTT and 1 µl of RNaseOUT were added and the mixture was incubated at 25ºC for 2 minutes in an MJ Research thermal cycler. This was stopped so that 1 µl of SuperscriptII enzyme could be added and then restarted for a further 10 minutes at 25ºC before shifting to 55ºC for 50 minutes. The reaction mixture was heat-inactivated by heating to 70ºC for 15 minutes, and RNA was removed by adding 1 µl of RNase H and incubating at 37ºC for 20 minutes. ;;Degenerate PCR reactions ;The procedure for this first round of degenerate PCR reaction on the cDNA derived from hybridoma cells was based on the method described by Wang et al. (2000) and Co et al. (1992). The primers for this round (Table 2) contain restriction sites to facilitate cloning into pBluescriptII plasmids. ;;The PCR reaction component (Table 3) was mixed on ice in thin-walled PCR tubes and then transferred to an MJ Research thermal cycler that was preheated and held at 94ºC. ;The reaction was carried out using a program derived from Wang et al., 2000, as follows: ;;Name: Wang45 ;94ºC 3:00 min ;94ºC 0:15 sec ;45ºC 1:00 min ;72ºC 2:00 min ;Goto 229 times ;72ºC 6:00 min ;4ºC rest ;End ;;The PCR reaction mixture was then run on a 1% low-melting agarose gel, bands of 300 to 400 bp were excised, purified using Zymo DNA minicolumns and sent to Agentcourt biosciences for sequencing. The respective 5’ and 3’ PCR primers were used as sequencing primers to generate these 38SB19 variable region cDNAs from both directions. ;;Cloning of the 5’ end sequence ;Because the degenerate primers used to clone the 38SB19 variable region light and heavy chain cDNA sequences alter the 5’ sequences, additional sequencing attempts were necessary to decipher the complete sequences. The preliminary cDNA sequence from the method described above was used to search the NCBI IgBlast site (http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline sequences from which the 38SB19 sequence is derived. PCR primers were designed (Table 3) to anneal the leader sequence of the murine antibody so that a new PCR reaction could yield the complete variable region cDNA, unaltered by the PCR primers. The PCR reactions, band purification, and sequencing were performed as described above. ;;Peptide analysis for sequence confirmation ;The cDNA sequence information for the variable region was combined with the germline constant region sequence to obtain full-length antibody cDNA sequences. The molecular weights for the heavy and light chains were then calculated and compared to molecular weights obtained by LC/MS analyses of the murine 38SB19 antibody. ;;Table 4 provides the calculated masses from the cDNA sequence for 38SB19 LC and -HC together with the values measured by LC/MS. The molecular weight measurements are consistent with the cDNA sequence for both the 38SB19 heavy and light chains. ;;Example 5 ;Recombinant expression of hu38SB19 antibodies ;The variable region sequences for hu38SB19 were codon optimized and synthesized by Blue Heron Biotechnology. The sequences are flanked by restriction enzyme sites for cloning inframe with the respective constant sequences in both single-chain and tandem dual-chain mammalian expression plasmids. The light chain variable region is cloned into the EcoRI and BsiWI sites in both ps38SB19LCZv1.0 and ps38SB19v1.00 plasmids (Fig. 5A and 5C). The heavy chain variable region is cloned into the HindIII and Apa1 sites in both ps38SB19HCNv1.0 and ps38SB19v1.00 plasmids (Fig. 5B and 5C). These plasmids can be used to express hu38SB19 in either transient or stable transfection in mammalian cells. Similar expression vector constructs were used to produce other chimeric and humanized antibodies. ;;Transient transfections to express hu38SB19 in HE-293T cells were performed using CaPO4 reagents from BD biosciences. The provided protocols were slightly modified for enhanced expression yields. Briefly, 2 x 10 <6>HE-293T cells were plated on 10 cm tissue culture plates coated with polyethyleneimine (PEI) 24 hours before transfection. Transfection began by washing the cells with PBS and replacing the medium with 10 ml DMEM (Invitrogen) with 1% Ultra low IgG FBS (Hyclone). ;Solution A (10 µg DNA, 86.8 µl Ca <2+> solution, and up to 500 µl with H2O) was added dropwise to Solution B while vortexing. The mixture was incubated at RT for 1 min and 1 ml of the mixture was added dropwise to each 10 cm plate. Approximately 16 h after transfection, medium was replaced with 10 ml fresh DMEM with 1% Ultra low IgG FBS. Approximately 24 h later, 2 mM sodium butyrate was added to each 10 cm plate. ;The transfection was harvested 4 days later. ;;Supernatant was prepared for Protein A affinity chromatography by adding 1/10 volume in 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant was filtered through a 0.22 µm filter membrane and loaded onto a Protein A Sepharose column (HiTrap Protein A HP, 1 ml, Amersham Biosciences) that was equilibrated with binding buffer (PBS, pH 7.3). A Q-Sepharose precolumn (10 ml) was connected upstream of the Protein A column during sample loading to reduce contamination from cellular material such as DNA. After sample loading, the precolumn was removed and the Protein A column orientation was reversed for washing and elution. The column was washed with binding buffer until a stable baseline was obtained, with no absorbance at 280 nm. An antibody was eluted with 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 ml/min. Fractions of approximately 0.25 ml were pooled and neutralized by the addition of 1/10 volume of 1M Tris/HCl, pH 8.0. Peak fractions were dialyzed overnight twice against PBS and purified antibody quantitated by absorbance at OD280. Humanized and chimeric antibodies can also be purified using a Protein G column with slightly different procedures. ;;All of the chimeric and humanized anti-CD38 antibodies described were expressed and purified by procedures similar to those described above. ;;Example 6 ;Antibody-dependent, cell-mediated cytotoxicity (ADCC) activities of chimeric anti-CD38 antibodies ;Because some anti-CD38 antibodies are previously known to have ADCC and/or CDC activity as chimeric or humanized antibodies with human IgG1 constant regions (J. H. Ellis et al.1995, J Immunol, 155: 925-937; F. K. Stevenson et al. 1991, Blood, 77: 1071-1079; WO 2005/103083), the chimeric versions of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39, consisting of murine variable regions and the human IgG1/IgKappa constant region, were generated and tested for ADCC and/or CDC activity. CDC activities. ;;Ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31 and ch38SB39 were first tested for ADCC using Ramos cells as target cells and human natural killer (NK) cells as effector cells. A lactate dehydrogenase (LDH) release assay was used to measure cell lysis (R. L. Shields et al., 2001, J Biol Chem, 276: 6591-6604). ;;NK cells were first isolated from human blood (from a normal donor; purchased from Research Blood Components, Inc., Brighton, MA) using a modified protocol for the NK Isolation Kit II (Miltenyi Biotech). Blood was diluted 2-3-fold with Hansk balanced salt solution (HBSS). 25 ml of diluted blood was carefully layered over 25 ml of Ficoll Paque in a 50 ml conical tube and centrifuged at 400 g for 45 min at 19ºC. The peripheral blood mononuclear cells (PBMC) were collected from the interface, transferred to a new 50 ml conical tube and washed once with HBSS. The PBMC were resuspended in 2 ml of NK isolation buffer and then 500 µl of Biotin-Antibody Cocktail (from the NK isolation kit, 130-091-152, Miltenyi Biotech) was added to the cell suspension. This Biotin-Antibody Cocktail contains biotinylated antibodies that bind to lymphocytes, except for NK cells. The mixture was incubated at 4ºC for 10 min, and then 1.5 ml of NK isolation buffer (PBS, 0.1% BSA, 1 mM EDTA) and 1 ml of anti-biotin microbeads were added. The cell-antibody mixture was incubated for another 15 min. at 4ºC. Then the cells were washed once with 50 ml NK isolation buffer and resuspended in 3 ml NK isolation buffer. Then the MACS LS column (on the MACS separator, Miltenyi Biotech) was prewashed with 5 ml NK isolation buffer. The cell suspension was then loaded onto the LS column. The effluent (the fraction with unlabeled cells) was collected in a new 50 ml conical tube. The column was washed three times with 3 ml NK isolation buffer. The entire effluent was collected in the same tube and washed once with 50 ml NK isolation buffer. NK cells were loaded onto the plate in 30 ml RPMI-1640 supplemented with 5% fetal bovine serum, 50 µg/ml gentamycin. ;;Various concentrations of ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31, and ch38SB39 antibodies in RPMI-1640 medium supplemented with 0.1% BSA, 20 mM HEPES, pH 7.4, and 50 µg/ml gentamycin (referred to above as RHBP medium) were aliquoted (50 µl/well) into a round-bottom 96-well plate. The target Ramos cells were resuspended at 10 <6 >cells/ml in RHBP medium and added to each well (100 µl/well) containing antibody dilutions. The plate containing target cells and antibody dilutions was incubated for 30 minutes at 37ºC. NK cells (50 µl/well) were then added to the wells containing the target cells, typically at a ratio of 1 target cell per 3-6 NK cells. RHBP medium (50 µl/well) was added to the control wells containing NK cells. Also, 20 µl of tTriton X-100 solution (RPMI-1640 medium, 10% Triton X-100) was added to the 3 wells containing only target cells without antibody, to determine the maximum possible LDH release. The mixtures were incubated at 37ºC for 4 hours, then centrifuged for 10 minutes at 12 rpm, after which 100 µl of the supernatant was carefully transferred to a new flat-bottom 96-well plate. The LDH reaction mixture (100 µl/well) from the Cytotoxicity Detection Kit (Roche 1644 793) was added to each well and incubated at room temperature for 5-30 minutes. The optical density of the samples was measured at 490 nm (OD490). The percentage specific lysis of each sample was determined by assigning 100% lysis to the OD490 value of Triton X-100-treated samples, and 0% lysis to the OD490 value of the untreated control sample containing only target cells. The samples containing only NK cells gave negligible OD490 readings. ;;When tested with Ramos target cells and NK effector cells, chimeric anti-CD38 antibodies showed highly potent ADCC activities (Fig. 6). The EC50 values were estimated by a nonlinear regression method with sigmoid dose-response curves and found to be as follows: 0.0013 µg/ml for h38SB13, 0.0013 µg/ml for ch38SB18, 0.0018 µg/ml for ch38SB19, 0.0022 µg/ml for ch38SB30, 0.0012 µg/ml for ch38SB31, 0.1132 µg/ml for ch38SB39. Chimeric anti-CD38 antibodies also showed potent ADCC activity on LP-1 (DSMZ ACC 41) multiple myeloma cells (EC50 values: 0.00056 μg/ml for ch38SB18, 0.00034 μg/ml for ch38SB19, 0.00024 μg/ml for ch38SB31) (Fig. 7A). Ch38SB19 also effectively killed Daudi lymphoma cells (Fig. 7B), NALM-6 B-ALL cells (DSMZ ACC 128) (Fig. 8A) and MOLT-4 T-ALL cells (ATTC CRL-1582) (Fig. 8B) by ADCC, suggesting that anti-CD38 antibodies with unusually potent apoptotic activity also have potent ADCC activity against various tumor cells derived from different hematopoietic malignancies. Neither a non-binding IgG1 control antibody (rituximab, BiogenIdec) (Fig. 7A, 8A and 8B) nor mu38SB19 (Fig. 7B) had any significant ADCC activity in the same experiment. ;;Example 7 ;CDC activities of chimeric anti-CD38 antibodies ;The CDC activities of ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31 and ch38SB39 were measured based on a method modified from H. Gazzano-Santoro et al.1997, J. Immunol Methods, 202: 163-171. Human complement was lyophilized human complement serum (Sigma-Aldrich S1764) which was reconstituted with sterile, purified water as suggested by the manufacturer and then diluted 5-fold with RHBP medium immediately before the experiment. Target cells suspended at 10 <6 >cells/ml in RHBP medium were aliquoted into wells of a flat-bottom 96-well tissue culture plate (50 µl/well). ;Then, 50 µl of different concentrations (from 10 nM to 0.001 nM) of anti-CD38 antibodies in RHBP medium were added (one antibody per well), followed by 50 µl/well of complement solution. The plate was then incubated for 2 hours at 37ºC in a humidified atmosphere containing 5% CO2, after which 50 µl of 40% Alamar Blue reagent (Biosource DAL1100), diluted in RHBP (10% final value), was added to each well to measure cell viability. Alamar Blue follows the reduction ability of the viable cells. The plate was incubated for 5-18 hours at 37ºC before measuring the fluorescence (in relative fluorescence units, RFU) at 540/590 nm. The percentage of specific cell viability for each sample was determined by first correcting the experimental values for background fluorescence by subtracting the background RFU value (wells with only medium, no cells) from the RFU values for each sample, and then dividing the corrected RFU values by the corrected RFU value for untreated cell samples. ;;When the CDC activity of the chimeric anti-CD38 antibody samples was tested with Raji-IMG cells using human complement at a final dilution of 5%, the chimeric anti-CD38 antibodies showed very potent CDC activities (Fig. 9). Raji-IMG are cells derived from Raji cells (ATCC CCL-86) and express lower levels of the membrane complement inhibitors CD55 and CD59. The EC50 values were estimated by non-linear regression from the sigmoid dose-response curve shown in Fig. 8 and are as follows: 0.005 µg/ml ch38SB13, 0.0101 µg/ml for ch38SB18, 0.028 µg/ml, for ch38SB19, 0.020 µg/ml for ch38SB30, 0.010 µg/ml for ch38SB31 and 0.400 µg/ml for ch38SB39. Chimeric anti-CD38 antibodies also showed potent CDC activity against LP-1 multiple myeloma cells (EC50 value: 0.032 μg/ml for ch38SB18, 0.030 μg/ml for ch38SB19, 0.043 μg/ml for ch38SB31), while a non-binding, chimeric control IgG1 (rituximab, BiogenIdec) had no CDC activity (Fig. 10). When chimeric CD38 antibodies were tested on Daudi lymphoma cells, different anti-CD38 antibodies differed in their respective CDC activities (Fig. 11). While the specific viability of Daudi cells was less than 15% after their incubation with 1.25 µg/ml ch38SB19 in the presence of complement, there was only a marginal decrease in the specific viability of these cells after their incubation with ch38SB18 and ch38SB39 (1.25 µg/ml or higher concentration) in the presence of complement (the specific viability was 85% and 91%, respectively). Furthermore, only a moderate decrease in specific viability was observed when Daudi cells were incubated with 1.25 µg/ml or higher concentration of ch38SB13, ch38SB30 and ch38SB31 in the presence of complement (the specific viability was 65%, 45% and 53%, respectively). ;;Example 8 ;Binding affinity and apoptotic, ADCC and CDC activities of humanized anti-CD38 antibodies ;The two versions of humanized 38SB19 (hu38SB19 v1.00 and v1.20) and the chimeric 38SB19 showed similar binding affinity when tested with Ramos cells with KD values of 0.23 nM, 0.25 nM and 0.18 nM, respectively (Fig. 12A). The binding affinity of chimeric and humanized 38SB19 antibodies was also collected in a competition binding assay in which their ability to compete with the binding of biotinylated, murine 28SB19 antibody is measured. Biotin-labeled murine 38SB19 antibody (3 x 10 <-10 >M) were mixed with various concentrations of ch38SB10, hu29SB19 v1.00 or hu38SB19 v1.20. The antibody mixture was incubated with Ramos cells and the amount of biotinylated, murine 38SB29 bound to the cells was measured with FITC-conjugated streptavidin by FACS analysis. Hu38SB19 v1.00, hu38SB19 v1.29 and ch38SB19 competed equally well with the binding of biotinylated, murine 38SB19 (Fig. 12B), again indicating that the binding affinity is unaffected by humanization. When ch38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 (10 <-8 >to 10 <-11 >M) were compared for their ability to induce apoptosis of Daudi cells, they showed similar apoptotic activities (Fig.139. Furthermore, hu38SB10 v1.00 and v1.20 also showed similar ADCC to ch38SB19 in LP-1 cells (Fig.14) and similar CDC potencies to ch38SB19 in Raji-IMG and LP-1 cells (Fig.15). Hu38SB19 v1.00 also showed similar CDC activity to ch28SB19 in the T-cell acute lymphoblastic leukemia cell line DND-41 (DSMZ 525) (Fig.15). Hu38SB19 v1.00 was further tested for its ability to induce apoptosis in a diverse set of cell lines (Fig.16). Treatment with hu38SB10 v1.00 (10 <-8 >M) resulted in a dramatic increase in Annexin V-positive cells in the B-cell lymphoma cell lines SU-DHL-8 (DSMZ ACC 583) (from 7% in untreated control to 97% in hu38SB10-treated cells) and NU-DUL-1 (DSMZ ACC 589) (from 10% in untreated control to 37% in hu38SB19-treated cells) and the T-ALL cell line DND-41 (from 7% in untreated control to 69% in hu38SB19-treated cells). In addition, treatment with hu38SB10 v1.00 (10 <-8 >M) the proportion of Annexin V-positive cells in the B-cell lymphocytic leukemia cell line JVM-13 (DSMZ ACC 19) (from 8% in untreated control to 17% in hu38SB19-treated cells) and in the hairy cell leukemia cell line HX-1 (DSMZ ACC 301) (from 6% in untreated control to 10% in hu38SB19-treated cells). ;;Similarly, two versions of humanized 38SB31 (hu38SB31 v1.1 and v1.2) and the chimeric 38SB31 showed similar binding affinities when tested with Ramos cells with KD values of 0.13 nM, 0.11 nM and 0.12 nM, respectively. The binding affinities of chimeric and humanized 38SB31 antibodies were also compared in a competition binding assay as described above, and performed equally well. Hu38SB31 v1.1 was further tested for its ability to induce apoptosis in additional cell lines. The humanized antibody showed similar apoptotic activities as ch38SB31 against Ramos, Daudi, Molp-8 and SUÆ-DHL-8 cells. Furthermore, hu38SB31 v1.1 also showed similar ADCC and CDC activities as ch38SB31 in these cell lines. ;;Example 9 ;In vivo efficacy of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 The in vivo anti-tumor activities of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 were investigated in a survival human xenograft tumor model in immunodeficient mice (female CB.17 SCID), established with Ramos lymphoma cells. Female CB.17 SCID mice were inoculated with 2 x 10 <6 >Ramos cells in 0.1 ml serum-free medium through a lateral tail vein. 7 days after tumor cell inoculation, mice were randomized into 7 groups based on body weight. There were 10 mice per group, except for the 38SB31-treated group which had 6 mice, and the 38SB39-treated group which had 8 mice. Antibodies were administered intravenously to mice at a dose of 40 mg/kg, twice per week, for three consecutive weeks, starting 7 days after cell inoculation. Mice were sacrificed if one or both hind limbs were paralyzed, body weight loss was more than 20% from the pretreatment value, or the animal was too sick to access food and water. Treatment with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 significantly prolonged the survival of the mice, compared to the PBS-treated mice (Fig. 17). The median survival for PBS-treated mice was 22 days and that of the antibody-treated groups ranged from 28 to 33 days. ;;The in vivo anti-tumor activities of mu38SB19 and hu38SB19 were further investigated in additional human xenograft tumor models in immunodeficient mice. For a Daudi lymphoma survival model, SCID mice were inoculated with 5 x 10 <6 >Daudi cells in 0.1 ml serum-free medium through a lateral tail vein. The study was performed as described above. Treatment with either mu38SB19 or hu38SB19 significantly prolonged the survival of mice compared with that of PBS-treated mice (Fig. 18). The median survival for PBS-treated mice was 22 days, while the median survival for antibody-treated mice was 47 days. ;;For an NCI-H929 multiple myeloma tumor model, SCID mice were inoculated subcutaneously with 10 <7 >cells. When tumors were palpable on day 6, animals were randomized into groups of 10 according to body weight and antibody treatment was initiated. The Hu38SB19 antibody or a non-binding, chimeric IgG1 control antibody (rituximab, BiogenIdec) was administered intravenously to mice at a dose of 40 mg/kg, twice weekly, for three consecutive weeks. Tumor volume was monitored and animals were sacrificed if the tumor reached 2000 mm <3 >in size or became necrotic. PBS-treated group reached a mean tumor volume of 1000 mm <3 >on day 89, the chimeric IgG1 control antibody group on day 84 (Fig. 19). Treatment with hu38SB19 completely prevented tumor growth in all 10 animals. In contrast, only two animals in the PBS-treated group and three animals in the chimeric IgG1 control antibody group showed tumor regression. ;;For a MOL-8 multiple myeloma tumor model, SCID mice were inoculated subcutaneously with 10 <7 >cells. When tumors were palpable on day 4, animals were randomized into groups of 10 according to body weight and antibody treatment was initiated. The hu38SB19 and mu38SB19 antibodies or a chimeric IgG1 control antibody were administered intravenously to mice at a dose of 40 mg/kg, twice weekly, for three consecutive weeks. Tumor volume was monitored and animals were sacrificed if tumors reached 2000 mm <3 >in size, or became necrotic. The PBS-treated group reached a mean tumor volume of 500 mm <3>on day 22, the chimeric IgG1 control antibody group on day 23 (Fig. 20). None of the tumors in these groups showed regression. In contrast, treatment with hu38SB19 or mu38SB19 led to tumor regression in 8 of 10 and 6 of 10 animals, respectively. ;Tables ;Table 1: ;Mu38SB19 light chain framework surface residues and corresponding residues at the same Kabat position in human 1.69 antibody. The residues that are different and therefore changed in the hu38SB19 antibody are in the shaded boxes. The marked (*) residues are backmutated to the mu38SB19 residue in one or more hu38SB19 variants.

Table 1B:

Mu38SB19 heavy chain framework surface residues and corresponding residues at the same Kabat position in the human 1.69 antibody. Residues that are different and therefore changed in the hu38SB19 antibody are shaded in gray.

Table 2

Primers used for the degenerate PCR reactions are based on those of Wang et al., 2000, except for HindKL which is based on Co et al., 1992. Mixed bases are defined as follows: H=A+T+C, S=g+C, Y=C+T, K= G+T, M=A+C, R=A+g, W=A+T, V = A+C+G.

Table 3

Light and heavy chain PCR reaction mixtures for cloning the 38SB19 variable region cDNA sequences.

Table 4

5’ end murine leader sequence primers used for 38SB19 second round PCR reactions. The 3’ end primers are identical to those used in the first round reactions because they primed to the respective constant region sequences.

Table 5

cDNA calculated and LC/MS measured molecular weights for the murine 38SB19 antibody light and heavy chains.

Claims (3)

Patent claims 1. One or more polynucleotides encoding an antibody or epitope-binding fragment thereof, wherein the antibody or epitope-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID NO: 66 and a light chain variable region (VL) comprising SEQ ID NO: 62 or 64. 2. A recombinant vector comprising one or more polynucleotides according to claim 1. 3. Host cell comprising a vector according to claim 2.